detecting bacterial contamination using magneto-fluorescent nanosensors

Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors

In order to prepare solutions for reading in the magnetic relaxometry, 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 in 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 readings are less quantifiable, which is why the use of fluorescence emission 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 supernatant is discarded and the bacterial pellet is resuspended in PBS. Finally, the sample may be analyzed via fluorescence emission. The strength of the emission will be relative to the amount of nanosensors remaining in solution, and therefore, also to the amount of bacteria present.

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1. 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 in the magnetic relaxometer. Open the respective software, select the &#34;T2 relaxation&#34; setting, and press &#34;measure.&#34;</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>
	</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>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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</td>
			<td>Sera care</td>
			<td>5310-0352</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-E. coli O157:H7 antibody [P3C6</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</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</td>
			<td>Tecan</td>
			<td>Infinite M200 PRO</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Column</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>

Source: Shelby, T., Sulthana, S., McAfee, J., Banerjee, T., Santra, S. <a href="https://app.jove.com/v/55821">Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to detect bacterial contaminants in food and water samples using nanosensors. The target pathogenic bacteria are detected through a combination of magnetic relaxation and fluorescence emission modalities.

1. Rapid Detection of E. coli O157:H7 using MFnS

	Spike various PBS solutions (1X, pH 7.4, 300 &#181;L) with increasing amounts of the 10-6 bacterial ...

Begin by adding magneto-fluorescent nanosensors to the bacterial samples. These nanoparticles contain bacteria-specific antibodies and a fluorescent dye.
Incubate to allow interaction with bacteria, causing detectable changes in the magnetic properties of the solution.
Include a baseline nanosensor solution without bacteria as a control.
Transfer each solution to a magnetic relaxometer, where a constant magnetic field aligns the magnetic particles within the sample.
Apply a brief pulse to disrupt alignment temporarily.
The relaxometer measures how quickly the particles return to their original state, known as the T2 relaxation time.
Nanosensors clustering around bacteria increases T2 compared to the control, enabling quantification even at low bacterial concentrations.
Conversely, higher bacterial concentrations decrease nanosensor clustering, affecting T2 values.
For accurate quantification, perform fluorescence measurement.
Accordingly, centrifuge to isolate bacteria and bound nanosensors.
Resuspend the pellet for fluorescence measurement.
Upon excitation, the fluorescent nanosensors emit light, aiding in bacterial measurement.

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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.

Nanosensors للكشف عن حوزتي النشاط في فيفو لتشخيص موسع

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

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Bioengineering

Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings

Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant bacteria, novel antibacterial agents, are urgently needed. A bacteriophage-based biocontrol strategy is the therapeutic use of phages to control bacterial pathogens in agricultural settings. Phage biocontrol is increasingly accepted as a sustainable technology, effective at decontaminating foodborne pathogens. To ensure effective biocontrol outcomes, systematic screening of phage combinations against targeted bacteria under required environmental conditions is crucial. Antibacterial efficacy of phage cocktails may be affected by phage genera and combination, targeted bacterial strains, the multiplicity of infection, temperature, and time. To formulate a phage cocktail with superior efficacy, the proposed method was to systematically evaluate the effectiveness of individual phages and phage cocktails in killing foodborne bacterial pathogens under targeted conditions. Bacterial killing efficacy was monitored by measuring optical density at desired temperatures and durations. Superior phage efficacy was determined by complete inhibition of bacterial growth. The proposed method is a robust, evidence-based approach to facilitate formulating phage cocktails with superior antibacterial efficacy.

This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails.

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Bacterial pathogens continually challenge food safety systems worldwide. With increasing concerns about the emergence of heat- and sanitizer-resistant ...

Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors

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Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors