Name: fluorescent paper strips for the detection of diesel adulteration with smartphone read-out
Uploaded: 2018-11-09T12:30:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out at JoVE.com

The authors would like to acknowledge the BAM for funding through the focus area Analytical Sciences: https://www.bam.de/Navigation/EN/Topics/Analytical-Sciences/Rapid-Oil-Test/rapid-oil-test.html.

1. Fluorescent Dyes (Figure 1A)
<ol>
	<li>Purchase commercially available 4-DNS and 4-DNS-OH. 
	Note: 4-DNS-COOH is not commercially available and is prepared from 4-DNS-OH as described hereafter.</li>
	<li>Place 50 mg (0.16 mmol) of 2-[ethyl[4-[2-(4-nitrophenyl)ethenyl]phenyl]amino]ethanol, 2 mg (0.016 mmol) of 4-dimethylaminopyridine and 19.2 mg (0.192 mmol) of succinic anhydride in a 10 mL round bottom flask.</li>
	<li>Dissolve the reagents in 2 mL of dry dichloromethane under argon atmosphere.</li>
	<li>Add 11.6 &#181;L (0.08 mmol) of triethylamine and let the mixture react for 20 h.</li>
	<li>Monitor the reaction by thin layer chromatography until quantitative conversion of the starting materials (Rf = 0.61) into the product (Rf = 0.27) is indicated (hexane/EtOAc, 4/6, v/v)</li>
	<li>Add 2 mL of water to the mixture before acidification to pH 2 with acetic acid (approx. 10 &#181;L).</li>
	<li>Extract the mixture by performing two successive liquid-liquid extractions, with 10 mL of dichloromethane each time.</li>
	<li>Wash once the reunited organic phases with 10 mL of saturated NaCl (&#62; 359 g L&#8211;1).</li>
	<li>Dry the organic phases by adding Na2SO4 powder until some fine drying agent powder remains visible.</li>
	<li>Purify the crude product by flash silica column chromatography with petroleum ether:ethylacetate 1:9 as the eluent. 
	Note: The yield achieved were 49 mg (74%) of the desired product.</li>
	<li>Perform 1H NMR analysis of the purified product in DMSO-d6 to validate the structure (&#948; 8.17 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 16.3 Hz, 1H), 7.10 (d, J = 16.3 Hz, 1H), 6.75 (d, J = 8.9 Hz, 2H), 4.18 (t, J = 6.0 Hz, 2H), 3.58 (t, J = 6.0 Hz, 2H), 3.43 (q, J = 7.0 Hz, 2H), 2.50 &#8211; 2.45 (m, 4H), 1.10 (t, J = 7.0 Hz, 3H) ppm).</li>
	<li>Perform 13C NMR analysis of the purified product in DMSO-d6 to validate the structure (&#948; 173.36, 172.20, 147.99, 145.23, 145.13, 133.89, 128.76, 126.30, 124.03, 123.67, 120.95, 111.58, 61.52, 48.05, 44.57, 28.73, 28.63, 12.00 ppm).</li>
	<li>Perform high resolution mass spectrometry with positive electro spray ionization of the purified product, corresponding to the calculated value (C22H25N2O6 [M+H]+: 413.1707) m/z ratio of 413.1713.</li>
</ol>
2. Synthesis of the Reference Dye
Note: The synthetic procedure of 8-(phenyl)-1,3,5,7-tetramethyl-2,6-diethyl-4,4-difluoro-4 bora-3a,4a-diaza-s-indacene was adopted from Coskun et al.18.
<ol>
	<li>Purify the crude product by column chromatography on silica with toluene as eluent. 
	Note: The yield achieved were 441 mg (29 %) of bright reddish crystals.</li>
	<li>Perform 1H NMR analysis of the pure product at 600 MHz in DMSO-d6 to validate the structure (&#948; 0.98 (t, 6H, J = 7.6 Hz), 1.27 (s, 6H), 2.29 (q, 4H, J = 7.6 Hz), 2.53 (s, 6H), 7.27-7.29 (m, 2H), 7.46-7.48 (m, 3H) ppm).</li>
	<li>Perform high resolution mass spectrometry with positive electro spray ionization of the purified product, corresponding to the calculated value (C23H28BF2N2 [M+H]+: 381.2314) m/z ratio of 381.2267.</li>
</ol>
3. TEST STRIP FABRICATION, METHOD 1.
<ol>
	<li>Prepare 1 mM solutions of the reference dye and dyes 4-DNS, 4-DNS-OH and 4-DNS-COOH in toluene.</li>
	<li>Cut cellulose strips of 30 &#215; 5 mm from filter paper.</li>
	<li>Place approximately 50 of those strips (611 mg) in a sealable 5 mL vial together with 4.5 mL of the desired dye solution from step 3.1.</li>
	<li>Shake the strips inside the vial with a vertical rotator for 20 min at 30 rpm.</li>
	<li>Pour the toluene solution out of the vial, and immediately fill with 4 mL of cyclohexane and rotate for 1 min at 30 rpm to wash off excess dyes.</li>
	<li>Repeat the washing operation from Step 3.5 three times.</li>
	<li>Dry the obtained test strips on a filter paper for 10 min in air at room temperature.</li>
</ol>
4. Test Strip Fabrication, Method 2.
<ol>
	<li>Amination of the paper strips.
	<ol>
		<li>Cut cellulose strips of 30 &#215; 5 mm from filter paper.</li>
		<li>Under a fume hood, place approximately 20 of those strips (308 mg) in a flask containing 40 mL of toluene.</li>
		<li>Add 960 &#181;L of 3-aminopropyltriethoxysilane (APTES) in the flask and stir the mixture for 24 h at 80 &#176;C.</li>
		<li>Remove the strips from the flask and wash thoroughly with 50 mL of ethanol.</li>
		<li>Dry the strips for 2 h at 50 &#176;C.</li>
	</ol>
	</li>
	<li>Grafting of the dye.
	<ol>
		<li>Under a fume hood, dissolve 5 mg of 4-DNS-COOH (13 &#181;mol) in 10 mL of dry dichloromethane under argon atmosphere in a 25 mL flask.</li>
		<li>Add N,N&#39;-dicyclohexylcarbodiimide (DCC, 3.3 mg, 16 &#181;mol) and allow the carboxylic acid to be activated for 15 min.</li>
		<li>Add triethylamine (2.2 &#181;L, 16 &#181;mol) and 18 aminated paper strips (278 mg).</li>
		<li>Stir the mixture for additional 2 h.</li>
		<li>Remove the strips from the solution and wash with 25 mL of dichloromethane and 25 mL of ethanol.</li>
	</ol>
	</li>
</ol>
5. Sample Pre-Treatment.
<ol>
	<li>Laboratory treatment
	<ol>
		<li>Place 10 mL of a fresh diesel/kerosene blend in a 25 mL vial.</li>
		<li>Suspend 10 wt% of active charcoal in the blend.</li>
		<li>Stir the vial for 1 h, centrifuge (400 x g, 10 min) and filter to remove the charcoal.</li>
	</ol>
	</li>
	<li>On-site treatment
	<ol>
		<li>Purchase circular activated carbon loaded filters of 47 mm diameter.</li>
		<li>Place four of the filters in a 47 mm stainless steel in-line filter holder.</li>
		<li>Flush 5 mL of a fresh diesel/kerosene blend through the filters with a standard 10 mL syringe; approximately 2 mL of polycyclic aromatic hydrocarbon-free solution was obtained.</li>
	</ol>
	</li>
</ol>
6. Smartphone Reader Implementation
Note: An Android based smartphone with a centered front camera was used as the core of the smartphone measurement system. All the necessary optical elements and 3D-printed accessory were custom-made for this device. However, any other smartphone with a CMOS (Complementary Metal Oxide Semiconductor) camera can be used.19,20
<ol>
	<li>Purchase a standard 5 mm epoxy LED at 460 nm, a 100 &#937; resistor and a USB on-the-go (OTG) cable with an ON/OFF switch and a micro USB port.</li>
	<li>Cut the USB cable on the opposite of the OTG side to isolate the red wire powering +5 V (up to 300 mA) and the black wire corresponding to the ground.</li>
	<li>Cut the black wire of the USB cable and solder the 100 &#937; resistor on the back of the switch. Solder the LED anode to the +5V red wire and the LED cathode to the ground black wire.</li>
	<li>Purchase a diffuser and two filters for the LED and the camera, typically a short pass filter for the excitation channel (LED) and a band pass filter for the emission collection (camera).</li>
	<li>3D-print a smartphone case that fits on the smartphone and integrates the different optical parts consisting of a black chamber (20 x 30 x 40 mm)21 as described in Figure 2.</li>
	<li>3D-print a strip holder as described in Figure 2 to hold a reference and a test strip.</li>
	<li>Implement the excitation channel by placing the LED, the diffuser and the filter to illuminate the paper strips at an angle of 60&#176;.</li>
	<li>Implement the reading channel by placing the filter in front of the smartphone CMOS camera.</li>
	<li>Insert the test strip holder containing the strips to start a measurement.</li>
</ol>
7. Sample Analysis Using the Smartphone-Based Detector
Note: Analyses were carried out by running a Java app(lication) for Android that finally displayed the adulteration level on the screen. Without the app, pictures can be taken, exported to a computer and analyzed with a standard image analysis software.
<ol>
	<li>Select the adequate calibration file, here diesel/kerosene, from the software memory by clicking on the Menu button in the upper-right corner of the software window.</li>
	<li>Dip the test strip into the diesel sample for a couple of seconds by holding the test strip with tweezers.</li>
	<li>Remove excess fuel by simple patting with a drying paper.</li>
	<li>Place the test strip inside the strip holder besides the reference strip and introduce the holder into the smartphone case. 
	Note: An image of the strips&#8217; fluorescence is then immediately displayed on the smartphone&#8217;s screen.</li>
	<li>Press the SHOOT button to record the fluorescence intensities of test and reference strips. 
	Note: The degree of adulteration is immediately calculated by the internal algorithm and displayed on the screen.</li>
</ol>

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The authors have nothing to disclose.

A fluorescent probe, based on a molecular rotor dye that is sensitive to viscosities in the range of those measured for diesel and its different blends with kerosene, was used to obtain simple and efficient test strips for the detection of diesel fuel adulteration. The emission intensity of 4-DNS at 550 nm in various diesel/kerosene blends correlates with a reduction in viscosity when the proportion of kerosene increases. At a temperature of 24 &#176;C, a nonlinear fluorescence quenching of up to 55% was observed for up ...

Fuel adulteration is a serious problem in many different parts of the world, simply due to the enormous relevance of fuel as an energy source. Running engines on adulterated fuel reduces their performance, leads to earlier engine failure and entails environmental pollution1. Increased SOx emissions occur if diesel is adulterated with kerosene that usually contains a higher amount of sulfur2,3. Although the problem exists for decades, sustainable fuel management that uncovers such criminal activity at its point of origin is still rare, because simple and reliable tests for fuel adulteration are largely lacking4. Despite substantial progress in laboratory-based mineral oil analysis in the past decades5,6,7, approaches to on-site measurements are still scarce. Various methods for the use outside of the laboratory have recently been devised, using fiber optics8, field-effect transistors9 or mechano-chromic materials10. Although they overcome some of the drawbacks of conventional methods, robust, user-friendly and portable methods are still lacking largely. Fluorescent viscosity probes based on molecular rotors are an interesting alternative11,12, because mineral oils are comprised of a great variety of hydrocarbons that differ in chain length and cyclicity, being often reflected in different viscosities. Because fuels are complex mixtures without specific lead compounds to act as tracers, the measurement of the change of a macroscopic property like viscosity or polarity seems very promising. The latter can be addressed by fluorescent molecular rotors for which the fluorescence quantum yields depend on environmental viscosity. After photoexcitation, deactivation commonly involves a twisted intramolecular charge transfer (TICT) state, the population of which is determined by the viscosity of its surrounding microenvironment13. Highly viscous solvents hinder molecular rotors to adopt a TICT state, entailing bright emission. In low-viscous solvents, the rotor can much better access the TICT state, accelerating non-radiative decay and thus quenched fluorescence. The addition of kerosene, with a viscosity of 1.64 mm2&#8729;s-1 at 27 &#176;C, to diesel, with respective viscosities of 1.3-2.4, 1.9-4.1, 2.0-4.5 or 5.5-24.0 mm2&#8729;s -1 at 40 &#176;C for grades 1D, 2D, EN 950 and 4D14,15,16, reduces the kinematic viscosity of the mixture and potentially leads to a proportional quenching of the fluorescence of a molecular rotor probe. The family of 4-dimethylamino-4-nitrostilbenes (4-DNS) seemed most promising to us because of their strong fluorescence variation over a kinematic viscosity range of 0.74-70.6 mm2&#8729;s -1. This range matches well with the known values of kerosene and diesel.
We therefore explored the ability of 4DNS, 2-[ethyl[4-[2-(4-nitrophenyl)ethenyl]phenyl]amino]ethanol (4DNSOH) and (E)-4-(2-(ethyl(4-(4-nitrostyryl)phenyl)amino)ethoxy)-4-oxobutanoic acid (4DNSCOOH) to indicate the viscosity of diesel-kerosene mixtures through their fluorescence, depending on intramolecular rotation and finally yielding a rapid test for diesel adulteration with kerosene. The disposable test is easy to use, precise, reliable, cost-effective and dimensionally small. The adsorption of the probes onto the filter paper as a solid support was investigated and the analysis was accomplished with an embedded smartphone-based fluorescence reader. Today, ubiquitously available smartphones are equipped with high-quality cameras, rendering the detection of optical changes such as color and fluorescence straightforward, and paving the way for powerful on-site analyses. We demonstrate here that the measurement of the emission of fluorescent probes adsorbed on paper strips with a smartphone can be used for fraud detection on combustion fuels in a reliable manner17.

<table><tbody><tr><td>4-dimethylamino-4-nitrostilbene (CAS Number: 2844-15-7)</td><td>Sigma-Aldrich</td><td>39255</td><td>4-DNS Dye</td></tr><tr><td>2-[ethyl[4-[2-(4-nitrophenyl)ethenyl]phenyl]amino]ethanol (CAS Number: 122258-56-4)</td><td>Sigma-Aldrich</td><td>518565</td><td>4-DNS-OH Dye</td></tr><tr><td>Whatman&amp;nbsp;qualitative filter paper, Grade 1</td><td>Sigma-Aldrich</td><td>Z274852</td><td>Test strips support</td></tr><tr><td>Whatman&amp;nbsp;application specific filter, activated carbon loaded paper, Grade 72</td><td>Sigma-Aldrich</td><td>WHA1872047</td><td>Fuel pre-treatment filters</td></tr><tr><td>Pall reusable in-line filter holders stainless steel, diam. 47&amp;nbsp;mm</td><td>Sigma-Aldrich</td><td>Z268453&amp;nbsp;</td><td>Holder pre-treatment filters</td></tr><tr><td>(3-Aminopropyl)triethoxysilane</td><td>Sigma-Aldrich</td><td>919-30-2</td><td>APTES</td></tr><tr><td>4-(Dimethylamino)pyridine</td><td>Sigma-Aldrich</td><td>1122-58-3</td><td>DMAP</td></tr><tr><td>Succinic anhydride</td><td>Sigma-Aldrich</td><td>108-30-5</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma-Aldrich</td><td>121-44-8</td><td>Et&lt;sub&gt;3&lt;/sub&gt;N</td></tr><tr><td>&lt;em&gt;N,N&#039;&lt;/em&gt;-dicyclohexylcarbodiimide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>538-75-0</td><td>DCC</td></tr><tr><td>Stuart Tube Rotators</td><td>Cole-Parmer</td><td>SB3</td><td>Rotator</td></tr><tr><td>FreeCAD</td><td>freecadweb.org</td><td>-</td><td>Freeware - 3D design</td></tr><tr><td>Ultimaker Cura</td><td>Ultimaker</td><td>-</td><td>Freeware - 3D printing</td></tr><tr><td>Android Studio</td><td>Google</td><td>-</td><td>Freeware - App programming</td></tr><tr><td>Renkforce SuperSoft OTG-Mirror Micro-USB Cable 0,15 m</td><td>Conrad.de</td><td>1359890&amp;nbsp;- 62</td><td>Smartphone setup electronic part</td></tr><tr><td>Black Cord Switch 1 x Off / On</td><td>Conrad.de</td><td>1371835&amp;nbsp;- 62</td><td>Smartphone setup electronic part</td></tr><tr><td>Carbon Film Resistor 100 &amp;Omega;</td><td>Conrad.de</td><td>1417639&amp;nbsp;- 62</td><td>Smartphone setup electronic part</td></tr><tr><td>492 nm blocking edge BrightLine&amp;nbsp;short-pass filter</td><td>Semrock</td><td>FF01-492/SP-25</td><td>Filter excitation</td></tr><tr><td>550/49&amp;nbsp;nm BrightLine single-band bandpass filter</td><td>Semrock</td><td>FF01-550/49-25</td><td>Filter emission</td></tr><tr><td>&amp;Oslash;1/2&quot; Unmounted N-BK7 Ground Glass Diffuser, 220 Grit</td><td>Thorlabs</td><td>DG05-220</td><td>Diffuser excitation</td></tr><tr><td>LED 465 nm, 9 cd, 20 mA, &amp;plusmn;15&amp;deg;, 5 mm clear epoxy</td><td>Roithner</td><td>RLS-B465</td><td>LED excitation</td></tr></tbody></table>

fluorescent paper strips for the detection of diesel adulteration with smartphone read-out

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate the content of kerosene in diesel/kerosene blends, a wide-spread activity to adulterate fuel. In solvents with low viscosity, the dyes rapidly deactivate via a so-called twisted intramolecular charge transfer state, efficiently quenching the fluorescence. Measurements of diesel/kerosene blends revealed a good linear correlation between the decrease in fluorescence and the increase of the fraction of the less viscous kerosene in diesel/kerosene blends. Immobilization of the hydroxy derivative 4-DNS-OH in cellulose paper yielded test strips that preserve the fluorescent indicator&#39;s behavior. Combination of the strips with a reader based on a smartphone and a controlling app allowed to create a simple field test. The method can reliably detect the presence of kerosene in diesel from 7 to 100%, outperforming present standard methods for diesel adulteration.

The three structures of the two commercial dyes 4-DNS and 4-DNS-OH and the synthesized dye 4-DNS-COOH contain a stilbene core element substituted with a donor (-NR2) and an acceptor (-NO2) group at both ends, the central double bond constituting the hinge of the so called &#39;molecular rotor&#39; (Figure 1A). The structures differ in amino group substitution pattern with short alkyl groups for 4-DNS, two slightly longer groups including...

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Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

Three fluorescent molecular rotors of 4-dimethylamino-4-nitrostilbene (4-DNS) were investigated for their potential use as viscosity probes to indicate ...

fluorescent-paper-strips-for-detection-diesel-adulteration-with

Research

JoVE Journal

Environment

9.3K Views.  Bundesanstalt für Materialforschung und –prüfung (BAM). Here, we present a protocol to detect the adulteration of diesel with kerosene using test strips coated with a fluorescent viscosity probe together with a smartphone-based analysis system.

Video: Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Biochemistry

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 ...

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 ...

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

Chemistry

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengineering

Iridium Oxide-reduced Graphene Oxide Nanohybrid Thin Film Modified Screen-printed Electrodes as Disposable Electrochemical Paper Microfluidic pH Sensors

A facile, controllable, inexpensive and green electrochemical synthesis of IrO2-graphene nanohybrid thin films is developed to fabricate an easy-to-use integrated paper microfluidic electrochemical pH sensor for resource-limited settings. Taking advantages from both pH meters and strips, the pH sensing platform is composed of hydrophobic barrier-patterned paper micropad (&#181;PAD) using polydimethylsiloxane (PDMS), screen-printed electrode (SPE) modified with IrO2-graphene films and molded acrylonitrile butadiene styrene (ABS) plastic holder. Repetitive cathodic potential cycling was employed for graphene oxide (GO) reduction which can completely remove electrochemically unstable oxygenated groups and generate a 2D defect-free homogeneous graphene thin film with excellent stability and electronic properties. A uniform and smooth IrO2 film in nanoscale grain size is anodically electrodeposited onto the graphene film, without any observable cracks. The resulting IrO2-RGO electrode showed slightly super-Nernstian responses from pH 2-12 in Britton-Robinson (B-R) buffers with good linearity, small hysteresis, low response time and reproducibility in different buffers, as well as low sensitivities to different interfering ionic species and dissolved oxygen. A simple portable digital pH meter is fabricated, whose signal is measured with a multimeter, using high input-impedance operational amplifier and consumer batteries. The pH values measured with the portable electrochemical paper-microfluidic pH sensors were consistent with those measured using a commercial laboratory pH meter with a glass electrode.

The study demonstrates the growth of iridium oxide-reduced graphene oxide (IrO2-RGO) nanohybrid thin films on irregular and rough screen-printed carbon substrate through a green electrochemical synthesis, and their implementation as a pH sensor with a patterned paper-fluidic platform.

A facile, controllable, inexpensive and green electrochemical synthesis of IrO2-graphene nanohybrid thin films is developed to fabricate an easy-to-use ...

Engineering

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.

Author Spotlight: High-Quality Quantum Dot Nanobeads for Sensitive Fluorescent Lateral Flow Immunoassays

Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB) and the detection of disease biomarkers using QDNB-based lateral flow immunoassay strips. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent strip reader within 15 min.

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...

Low-Cost, Volume-Controlled Dipstick Urinalysis for Home-Testing

Dipstick urinalysis provides quick and affordable estimations of multiple physiological conditions but requires good technique and training to use accurately. Manual performance of dipstick urinalysis relies on good human color vision, proper lighting control, and error-prone, time-sensitive comparisons to chart colors. By automating the key steps in the dipstick urinalysis test, potential sources of error can be eliminated, allowing self-testing at home. We describe the steps necessary to create a customizable device to perform automated urinalysis testing in any environment. The device is cheap to manufacture and simple to assemble. We describe the key steps involved in customizing it for the dipstick of choice and for customizing a mobile phone app to analyze the results. We demonstrate its use to perform urinalysis and discuss the critical measurements and fabrication steps necessary to ensure robust operation. We then compare the proposed method to the dip-and-wipe method, the gold standard technique for dipstick urinalysis.

Dipstick urinalysis is a quick and affordable method of assessing one&#8217;s personal state of health. We present a method to perform accurate, low-cost dipstick urinalysis that removes the primary sources of error associated with traditional dip-and-wipe protocols and is simple enough to be performed by lay users at home.

Dipstick urinalysis provides quick and affordable estimations of multiple physiological conditions but requires good technique and training to use ...

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Fluorescent Paper Strips for the Detection of Diesel Adulteration with Smartphone Read-out

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