This study was funded in part by National Natural Science Foundation of China Grants (31370813, 3147670, 31670831 and 31600672,), the National Transgenic Plant Special Fund (2016ZX08012-003, 2016ZX08012-005), Program for New Century Excellent Talents in University, the National Key Research and Development Project of China (2016YFA0500601) and China Postdoctoral Science Foundation (2016M591667).

NOTE: This protocol assumes that the stainless steel mold bearing the shape for the desired micro-channels and the loading adaptor have already been made (3D files are provided as Supplemental Files 1 and 2). This protocol also assumes that plant DNA isolation has already been carried out.
1. Fabrication of the Capillary Array-based Ready-to-use Cassette
<ol>
	<li>Clean the stainless-steel mold.
	<ol>
		<li>Wash the stainless-steel mold by detergent, ethanol, and deionized water to remove the potential contaminants. Dry the mold by nitrogen.</li>
	</ol>
	</li>
	<li>Clean the capillaries
	<ol>
		<li>Wear safety goggles, lab&#160;uniform, and chemical protective gloves.</li>
		<li>Place capillaries into a beaker. Clean the capillaries with 30 mL acetone for 5 min to remove organic matter, and then wash the capillaries with deionized water. 
		Caution: Handle acetone in a fume hood.</li>
		<li>Pour 10 mL of H2O2 into the beaker&#160;and then add 30 mL of H2SO4 into the H2O2 with slow shaking to prevent overheating. Make sure that the solution (piranha solution) completely covers the capillaries for at least 30 min. 
		Caution: Be careful while handling piranha solution (H2SO4/H2O2 = 3: 1, v/v). If the piranha solution is spilled, wash away the solution quickly with a large amount of water and wipe the surface with paper towels. 
		NOTE: Thorough cleaning of capillaries is one of the key points to ensure the success of the LAMP reaction. It is necessary to shake the acid cylinder during the cleaning process to remove the bubbles in capillaries, and the clean time can also be extended when required.</li>
		<li>Wash away the piranha solution with a large amount of water.Wash capillaries with ethanol and deionized water for 5 min each. Dry the capillaries in a drying oven.</li>
	</ol>
	</li>
	<li>Pour polydimethylsiloxane (PDMS)
	<ol>
		<li>Weight out PDMS base and curing reagents with a ratio of 1:1 in a 50 mL tube. Typically, mix 5 g of elastomer base to 5 g of elastomer curing agent.</li>
		<li>Stir the mixture thoroughly for about 5 min with a glass rod. Place the tube with PDMS in a vacuum bell jar for 30 min for de-gassing.</li>
		<li>Pour the PDMS slowly into the cylinder of the stainless-steel mold.Allow the PDMS to cure for 3 h in a drying oven at 60 &#176;C 
		NOTE: Be careful to avoid air bubbles when pouring PDMS. After pouring PDMS, allow it stand for 5 min for natural removal of bubbles out of PDMS.</li>
	</ol>
	</li>
	<li>Remove the mold from PDMS
	<ol>
		<li>After curing the PDMS, remove the mold by pulling out the mold with cylinder. Remove PDMS from the cylinder by cutting the margin of mold with a scalpel.</li>
		<li>Wash PDMS three times with ethanol and deionized water. Dry the PDMS support with nitrogen.</li>
	</ol>
	</li>
	<li>Treat the lower surface of the PDMS support to be hydrophobic. Soak the PDMS support in a super-hydrophobic coat for 1 s. Dry it for 10 min at room temperature.</li>
	<li>Insert the capillary into the PDMS support
	<ol>
		<li>Insert the cleaned 4 mm capillaries into the holes of the PDMS support and leave 0.5 mm of the capillaries outside the top surface of the PDMS support. Make sure the ends of capillaries are on the same level.</li>
	</ol>
	</li>
	<li>Treat the outer surface of the capillaries and the top surface of the PDMS support to be hydrophobic.
	<ol>
		<li>Add 15 &#181;L super-hydrophobic coat onto the top surface of the PDMS support. Note that the coating immediately spreads all over the top surface (including the top surface of the PDMS support and the outer surfaces of the exposed part of the capillaries) by capillary force.</li>
		<li>Air dry the super-hydrophobic modified capillary array.</li>
	</ol>
	</li>
	<li>Fix primer into capillary array
	<ol>
		<li>Prepare primer solution. Weight out 0.65 g chitosan (molecular formula: (C6H11NO4)n) and dissolve it into 50 mL deionized water by adjusting the pH to 4.5-5.5 with acetic acid to achieve a chitosan concentration of 1.3%. Prepare primer components mix as per Table 1. See Supplementary Table 1 for primers</li>
		<li>Add 1.6 &#181;L of the mixture of one set of primers to fill one corresponding capillary of the capillary array according to a pre-designed order. 
		NOTE: The remaining two blank capillaries were set as negative controls.</li>
		<li>Anchor the array in a transparent well of a standard flat bottom 96-well plate and dry the array at 60 &#176;C for at least 2 h.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LAMP primer fixing mix components (initial concentration)</td>
			<td>Volume (&#956;L)</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>17.0</td>
		</tr>
		<tr>
			<td>Chitosan (1.3%)</td>
			<td>1.0</td>
		</tr>
		<tr>
			<td>FIP/BIP primer (20 &#956;M)</td>
			<td>2.0/2.0</td>
		</tr>
		<tr>
			<td>LoopF/LoopB primer (20 &#956;M)</td>
			<td>1.0/1.0</td>
		</tr>
		<tr>
			<td>F3/B3 primer (20 &#956;M)</td>
			<td>0.5/0.5</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>25.0</td>
		</tr>
	</tbody>
</table>
Table 1: The components of LAMP primer fixing reagents. The components of LAMP primer fixing mix are listed in the left column of the table, and the volume of each component is listed in the right column.
<ol start="9">
	<li>Assemble the cassette as follows. Put a loading adaptor onto the top of anchored array. Make sure the dish of the adaptor just covers the exposed parts of all ten capillaries (See Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56597/56597fig1.jpg" /> 
Figure 1: The cassette fabrication and assembly. (a) Stainless mold and the PDMS support. The mold consists of 3 parts: cylinder, dam board, and pillar plate. (b) The schematic of PDMS support fabrication and assembly of the capillary cassette. The whole process contains 5 steps: 1. PDMS pouring, 2. Mold removing, 3. Capillary inserting and surface coating, 4. primer fixing and 5. cassette anchoring. 1. Pour PDMS into cylinder of the mold; 2. Push out the dam board to remove the mold from PDMS support; 3. Coat the down surface of PDMS support and then insert the capillaries into the PDMS support, lastly coat the upper surface of PDMS support and exposed capillaries. The thick blue line indicates super-hydrophobic coat; 4. Load primer set into individual capillaries; 5. Anchor the capillary array in a single 96-well plate and install a sample loading adaptor onto it. The details have been described in protocol steps 1.1 - 1.9. <a href="//cloudfront.jove.com/files/ftp_upload/56597/56597fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Performance of LAMP Reaction in Capillary Array
<ol>
	<li>Prepare the reaction mix
	<ol>
		<li>Prepare the LAMP reaction mix as per Table 2.</li>
		<li>Add reagents to mixture according to the chosen order, as in Table 2, and vortex the reaction mixture for 5 s after adding calcein. Gently invert the tube 20 times after Bst polymerase is added.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>LAMP components&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (initial concentration)</td>
			<td>Volume (&#956;L)</td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>11.6</td>
		</tr>
		<tr>
			<td>MgSO4 (100 mM)</td>
			<td>2.0</td>
		</tr>
		<tr>
			<td>dNTPs (25 mM)</td>
			<td>1.4</td>
		</tr>
		<tr>
			<td>Betaine (5 M)</td>
			<td>4.0</td>
		</tr>
		<tr>
			<td>Buffer (10x)</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>Calcein (1.25 mM)</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>MnCl2 (25 mM)</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Bst polymerase (8 U/&#956;L)</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>plant DNA (10 ng/&#956;L)</td>
			<td>1.0</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>25.0</td>
		</tr>
	</tbody>
</table>
Table 2: The reaction system of the capillary array-LAMP. The components of the reaction system of capillary array LAMP components are listed in left column, and the volume of each component is listed in the right column.
<ol start="2">
	<li>Load the reagents and seal the cassette
	<ol>
		<li>Pipette 20 &#181;L LAMP reaction mixture with a standard 100 &#181;L tip. Insert the tip into the inlet of the loading adaptor to lock it. Gently inject the reaction mixture into the dish of adaptor; the reaction mixture will quickly fill the dish and then load the capillaries automatically through capillary force.</li>
		<li>Remove the adaptor with the locked tip and seal the well by a PCR-compatible transparent sealing film. 
		NOTE: Only the capillaries touching the reaction mixture in the hydrophilic dish could be filled. So make sure the top side of all the capillaries are of the same height (See Figure 2).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56597/56597fig2.jpg" /> 
Figure 2: Diagram of sample-loading using the loading adaptor. The picture shows the loading process employing blue solution as an example. Insert the tip into the inlet and inject the sample into the adaptor slowly, and then remove the adaptor with locked tip. <a href="//cloudfront.jove.com/files/ftp_upload/56597/56597fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Incubate the capillary array in an incubator at 63 &#176;C for 1 h.</li>
</ol>
3. Results Readout and Data Analysis
<ol>
	<li>Acquire images of the fluorescence emission.
	<ol>
		<li>Fix a UV filter on the top surface of a small hand-held UV LED flashlight to filter the visible light.</li>
		<li>Excite the dissociated calcein to emit fluorescence with the UV flashlight. Capture images from the top of the capillary array by either a digital camera or a smartphone.</li>
		<li>When taking an image, ensure that the camera is zoomed in on the area of the capillaries as much as possible to get high quality, clear images.</li>
	</ol>
	</li>
	<li>Analyze the results
	<ol>
		<li>Open the image by image analysis software and then select &#34;Image&#62;Mold&#62;Grayscale&#62;Yes&#34; and &#34;Image&#62;Mold&#62;16bit&#62;Yes&#34;. Select &#34;File&#62;Save as&#62;format&#62;TIFF&#34; to convert the image to 16-bit TIFF format.</li>
		<li>Extract the value of fluorescence intensity
		<ol>
			<li>Open the microarray analysis software. Drag the 16-bit TIFF format image into the interface of the software and then select the wavelength of &#34;532 nm&#34; and a color of &#34;green&#34; to display the image.</li>
			<li>Create a new block to locate the fluorescence signal from the capillaries. Select &#34;Tools&#62;New blocks&#34; and input the number of columns and rows as &#34;1;1&#34; and &#34;2;5&#34; in the &#39;blocks&#39; and &#39;features&#39; interfaces separately.</li>
			<li>Right click and select &#34;Features&#34; model and then adjust the location and diameter to fit the fluorescence area of capillaries. Select &#34;Analyze&#34; to extract the value of fluorescence intensity.</li>
			<li>Select &#34;File&#62;save settings as&#34; to save the blocks documents and select &#34;File&#62;save results as&#34; to save the fluorescence intensity results. Calculate the SNR to define whether LAMP is successfully performed in the capillaries. 
			NOTE: Signals of the capillaries were obtained by recording the gray values of the spots and signal to noise ratios (SNRs) were defined as ratios of mean values of foreground signals of the targets to average mean values of foreground signals of the two negative controls. The cutoff to determine positive signals was set as SNR &#62;2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+detection+using+recombination+proteins.">DNA detection using recombination proteins.</a> Plos Biol. 4 (7), 1115-1121 (2006).</li><li>Opota, O., Jaton, K., Greub, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+diagnosis+of+bloodstream+infection:+towards+molecular+diagnosis+directly+from+blood.">Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.</a> Clin Microbiol Infect. 21 (4), 323-331 (2015).</li></ol>

The authors have nothing to disclose.

The CALM platform demonstrated here, which combines the LAMP technology with a capillary array, enables the simultaneous detection of multiple GMO-related gene targets in a single, highly effective and easy to operate test.
To successfully perform the multiplex LAMP reactions in the cassette, three critical points need to be noticed. Firstly, achieving the same height for the upper side of the capillaries and the hydrophilic and hydrophobic pattern of the capillary array are critical for simul...

Low-cost, quick, and easy to use systems for the simultaneous detection of multiple nucleic acids are urgently needed in a wide range of fields, such as clinical diagnostics1,2,3, GMO detection4,5,6, microbial monitoring7,8,9, forensic analysis10,11, and especially point-of-care tests (POCTs), where resources are usually limited12,13,14.
Polymerase chain reaction (PCR), including its derivative methods real-time PCR and multiplex PCR, is the most widely applied technique for detection in these fields. However, these methods typically only detect one target in one test15 and they require electricity and sophisticated professional equipment.
Another promising technology for detecting nucleic acids is Loop-mediated isothermal amplification (LAMP), which was first described in 200016. LAMP is a high efficiency DNA detection method. Theoretically, it can amplify from 1 copy to 109 copies of amplicons within one hour, all performed at a constant temperature, (i.e., between 60 - 65 &#176;C). Successful amplification will produce a large amount of the insoluble byproduct pyrophosphate and cause a change in turbidity17, which could be directly observed by the naked eye. A color change can also be observed by the addition of metal ions or fluorescent dyes such as Calcein18, Nucleic acid dye19, and hydroxyl naphthol blue20. Because of the advantages of high sensitivity and convenience of operation, LAMP is being widely applied in nucleic acid detection.
Currently, there are mainly two strategies for multiplex LAMP assays. One is to perform multiple LAMP assays by having multiple LAMP primer sets in one tube21,22,23. However, the multiplicity and the amplification efficiency would be limited by the intrinsic interference and competition among different primer sets. Furthermore, it can be difficult to identify different LAMP products in the same reaction. Another strategy is based on physical isolation. Different primer sets were isolated into individual miniaturized compartments, and multiple LAMP reactions are then performed simultaneously24,25. These approaches, which are generally based on microfluidic chips, provide a potential solution for high-throughput LAMP reactions. However, the manufacture of the chips and the multiplex pre-coating of primer sets is complicated, which may increase costs and decrease reproducibility.
Recently, a few studies have described performing LAMP reactions in capillaries to bypass the complicated fabrication of microfluidic chips and have achieved low-cost detection26,27. However, with regards to high-throughput analysis, these capillaries are similar to miniature versions of PCR strip tubes, because the samples and reaction reagents (including the different primer sets) must be individually prepared and delivered to different reaction units within capillaries. To achieve parallel and multiplex analysis, additional equipment, for example a multichannel syringe pump, is required for parallel loading of samples or reagents.
To overcome the limitations associated with the current methods for multiplex detection of nucleic acids, we have developed a miniaturized platform which combines visual LAMP technology with a capillary array. This platform is multi-target, compact in size, low cost, and easy to operate28. Herein, we describe the details of how to fabricate the capillary array and perform the LAMP reactions in the array. The protocol described here has been standardized using genetically modified organism (GMO) detection as a model. Importantly, this protocol can also be used in high-throughput detection of other nucleic acid targets.

<table><tbody><tr><td>UltraEverDry(super-hydrophobic coat)</td><td>UltraTech</td><td>4001</td><td>supplier:Exiron chemistry(CHINA) CO.,LTD.</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>8332557</td><td></td></tr><tr><td>Bst polymerase</td><td>New England BioLabs</td><td>M0275L</td><td></td></tr><tr><td>betain</td><td>Sigma-Aldrich</td><td>B0300-1VL</td><td></td></tr><tr><td>calcein</td><td>Sigma-Aldrich</td><td>C0875-5G</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>MKBP0495V</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>New England BioLabs</td><td>B1003S</td><td></td></tr><tr><td>dNTPs</td><td>Shanghai Sangon</td><td>B804BA0022</td><td></td></tr><tr><td>chitosan</td><td>Shanghai Sangon</td><td>LJ0805S309J</td><td></td></tr><tr><td>Photoshop 7.0 software</td><td>Adobe Systems Inc., CA, USA</td><td></td><td>Image analysis</td></tr><tr><td>GenePix Pro 6.1</td><td>Molecular Devices, CA, USA</td><td></td><td>microarray analysis software</td></tr><tr><td>AutoCAD</td><td>Adobe Systems Inc.</td><td></td><td>3D construction software</td></tr><tr><td>UV filter (ZWB2)</td><td>YXSensing</td><td></td><td>supplier : taobao</td></tr></tbody></table>

visual detection of multiple nucleic acids in a capillary array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

In this method, it is important to prevent cross-contamination among different capillaries during the sample loading. For this purpose, chitosan was introduced, which could retain the primers in individual capillaries. To test whether it worked or not, we pre-fixed the ADH1 (endogenous reference gene of maize) primer set in the capillary cassette with the pattern of &#34;T&#34; and &#34;U&#34;, as illustrated in Figure 3a. As...

Watch this Scientific Journal Video about Visual Detection of Multiple Nucleic Acids in a Capillary Array at JoVE.com

Visual Detection of Multiple Nucleic Acids in a Capillary Array

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

visual-detection-of-multiple-nucleic-acids-in-a-capillary-array

Research

JoVE Journal

Bioengineering

7.2K Views.  Shanghai Jiao Tong University. Dit protocol beschrijft de fabricage van een kleine, kant-en-klare cassette die kan worden toegepast voor visuele detectie van meerdere nucleic zuren in een enkele, test die is eenvoudig te bedienen. In deze benadering werd een capillaire matrix gebruikt voor multiplex en zeer efficiënte opsporing van GGO-doelstellingen.

Video: Visual Detection of Multiple Nucleic Acids in a Capillary Array

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

Genetics

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Immunology and Infection

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

Biochemistry

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

Chemistry

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Precision DNAzyme Nanomachines for Discriminative RNA and DNA Analysis

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

Author Spotlight: Advancements in DNA Nanosensors &#8211; Addressing Sensitivity and Selectivity Challenges in Molecular Detection

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Biology

Capillary Electrophoresis: A Technique to Analyze Fluorescently Labeled Polymerase Chain Reaction Amplicons

Source: Gulla, S. et al., <a href="https://www.jove.com/v/59455">Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and Capillary Electrophoresis</a>. J. Vis. Exp. (2019)
This video describes the technique of capillary electrophoresis to analyze fluorescently labeled PCR amplicons obtained from various samples based on their size. It generates a migratory pattern that determines the fragments&#8217; base-pair lengths with the help of the labeled fluorescent dye in each fragment.

Source: Gulla, S. et al., Multi-locus Variable-number Tandem-repeat Analysis of the Fish-pathogenic Bacterium Yersinia ruckeri by Multiplex PCR and ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Chip-Based Digital PCR to Detect Rare Transcript Variants Using a Nanofluidic Chip

Source: Molinari, C., et al. <a href="https://app.jove.com/v/57066">Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR.</a> J. Vis. Exp. (2018).
This video demonstrates chip-based digital PCR &#8212; a variation of the digital PCR technique that is useful in detecting rare transcript variants. The PCR reaction is partitioned into the chambers of a nanofluidic chip, each of which acts as an independent reaction. The detection of fluorescence signals from the chambers with amplified targets confirms the presence of rare transcript variants in the sample.

Source: Molinari, C., et al. Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR. J. Vis. Exp. ...

PCR Techniques

Detecting Circulating Anti-Glycan Antibodies with a Printed Glycan Array

Source: Olivera-Ardid, S. et al., <a href="https://app.jove.com/v/57662">Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates printed glycan array technology for analyzing circulating anti-glycan antibodies in mouse serum. The antibodies in the serum interact with specific glycans on the chip, which is confirmed by immunostaining followed by fluorescence scanning.

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Samenvatting

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