Ministry of Science and Technology of Taiwan provided financial support of this research under contracts MOST-103-2221-E-002 -097 -MY3.

1. Method to Detect MNP
Note: This section describes the procedure to detect the MNP based on the hybridization-mediated growth of gold nanoparticles.
<ol>
	<li>Prepare the probe DNA (5&#39;-thiol-GAGCTGGTGGCGTAGGCAAG-3&#39;) solution at a concentration 100 &#181;M.</li>
	<li>Prepare the probe DNA-modified AuNP (AuNP probe) particles.21 
	Note: The volume used here depends on the requirement of probe DNA-modified AuNP (AuNP probe) particles. It is hence dependent on the number of experiments to b...

<ol>
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In this protocol, a simple colorimetric method to detect MNP can be implemented at concentrations ranging from 0.11-0.50 &#181;M in microcentrifuge tubes. Furthermore, the proposed MNP detection method is conducted on a pneumatic droplet manipulation platform that has a high potential for DNA screening and other bio-medical applications. In practice, the detectable range of the sample DNA concentration depends on the mixing efficiency of the operating platforms. To ensure that the coalesced droplet is fully mixed, the cr...

Single-nucleotide polymorphism (SNP), which is a single base-pair difference in a DNA sequence, is one of the most common genetic variations. Current studies report that SNPs are associated with disease risk, drug efficacy and side-effects of individuals by affecting gene function.1,2 Recent studies also revealed that two- or multi-point mutations (multi-nucleotide polymorphism) cause particular diseases and individual differences in the effects of disease.3,4 The detection of nucleotide polymorphism is therefore imperative in prescreening disease. Simple and efficient methods for the rapid detection of sequence-specific oligonucleotides were highly developed in the past two decades.1,5 Current approaches to identify DNA mutations typically involve procedures including probe immobilization, fluorescence labeling, gel electrophoresis, etc.,6,7 but those methods generally require a long analytical process, expensive equipment, well trained technicians, and significant consumption of samples and reagents.
A nanoparticle with a large ratio of surface area to volume and unique physicochemical properties is an ideal material as a highly sensitive and cheap detection platform for specific biomarkers. Gold nanoparticles (AuNP) are widely used for DNA detection because of their great ability to be modified with oligonucleotide probes.8-10 SNP detection techniques were also developed using AuNP.11-13 In this work we adopted a novel colorimetric approach to detect the multi-nucleotide polymorphism (MNP) through DNA hybridization-mediated growth of AuNP probes.14 This simple and rapid probing method is based on the theory that varied lengths of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) conjugated to AuNP influence the growth size and shape of the AuNP (see Figure 1).15 This method of DNA detection features a small consumption of reagents, a small assay duration (a few minutes), and a simple procedure without thermal control that is prospectively applicable for clinical diagnosis and domestic medical screening.
Several microfluidic systems to detect the DNA sequence have been developed;16 those microfluidic systems, evolved from traditional experimental protocols, required fewer pieces of large-scale equipment and simplified the experimental protocols so as to improve the sensitivity, detection limit and specificity of the DNA biosensor. DNA detection methods in the microfluidic systems still require, however, instruments of a subsequent process such as a PCR (polymerase chain reaction) machine for signal amplification and a fluorescence reader for a single readout to identify the heterogeneous SNP.17,18 Developing a simple platform without the subsequent processing to directly read out the results of multi-nucleotide polymorphism is highly desirable. Compared to well used, conventional, closed microfluidic systems, the open-surface microfluidic devices promisingly offer several advantages, such as a clear optical path, an easy way to access the sample, a direct environmental accessibility and no easily formed cavitation or interfacial obstruction in the channel.19 Our previous work introduced a simple pneumatic platform for open-surface droplet manipulation (see Figure 2).20 On this platform, droplets can be simultaneously transported and manipulated without interference from a driving energy using a suction force, which has a great potential in biological and chemical applications. This pneumatic platform was thus utilized to execute the manipulation of DNA samples for MNP detection in combination with the colorimetric approach using the concept of DNA hybridization-mediated growth of AuNP probes.
The protocol presented in this paper describes a simple visual detection of multi-nucleotide polymorphisms on the pneumatic droplet manipulation platform on an open surface. This work confirms that multi-nucleotide polymorphism is detectable with the naked eye; the proposed pneumatic platform is suitable for biological and chemical applications.

<table border="0" cellpadding="0" cellspacing="0" width="735">
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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">PDMS</td>
			<td style="width:204px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">benchtop engravers</td>
			<td style="width:204px;">&#160;Roland DG</td>
			<td style="width:119px;">EGX-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">laser cutting machine</td>
			<td style="width:204px;">Universal Laser Systems, Inc.</td>
			<td style="width:119px;">VLS 3.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Oxygen plasma treatment system</td>
			<td style="width:204px;">Femto Science Inc. Korea</td>
			<td style="width:119px;">CUTE-MPR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">solenoid valve</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td>home built</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">vacuum pump</td>
			<td>ULVAC KIKO, Inc.</td>
			<td style="width:119px;">DA-30D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">13-nm AuNP solution</td>
			<td style="width:204px;">TAN Bead Inc., Taiwan</td>
			<td style="width:119px;">NG-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNA (with 5 -end labeled thiol)</td>
			<td style="width:204px;">MDBio, Inc., Taiwan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">phosphate buffered saline (PBS)</td>
			<td>UniRegion&#160;Bio-Tech,. Taiwan</td>
			<td>PBS001-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium dodecyl sulfate (SDS)</td>
			<td style="width:204px;">J. T. baker</td>
			<td style="width:119px;">4095-04</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Hydroxylamine solution (NH2OH)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:119px;">467804</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Chloroauric acid (HAuCl4)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>G4022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium chloride (NaCl)</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">vortex mixer</td>
			<td style="width:204px;">Digisystem Laboratory Instruments Inc.</td>
			<td style="width:119px;">VM-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">centrifuge</td>
			<td style="width:204px;">Hermle Labortechnik GmbH.</td>
			<td style="width:119px;">Z 216 MK</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this work, three DNA samples were tested using a simple and novel method of detection through the DNA hybridization-mediated growth of the AuNP probes. The sequences of probe DNA and DNA samples of three kinds, specifically, CDNA (fully complementary to probe DNA), TMDNA (three base-pair mismatched DNA), and SixMDNA (six base-pair mismatched DNA) are listed in Protocol step 1. The mismatches to the probe of the DNA samples tested here are both in the middle segments of the DNA samples....

Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

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

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. ...

The overall goal of this procedure is to combine a simple method for visually detecting multi-nucleotide polymorphisms with a pneumatic droplet manipulation platform. The DNA detection platform is on an open surface and is straight forward to use for biochemical researchers. We investigated a simple tool to repeatedly detect genetic mutations and genetic diseases.It has greater potential helping us understand how genetic variation leads to disease. We combined two novel and simple methods. The multi-nucleotide polymorphism can be detected on the pneumatic droplet manipulation platform with the naked eye and without any additional instrument.The method for detecting multi-nucleotide polymorphism will be demonstrated by Tzu-Ming Wang, the post doctor in our group. To begin this procedure, prepare a 100 micromolar solution of probe DNA as indicated in the text protocol. Then, add the probe DNA to a one OD per milliliter solution of gold nanoparticles.Bring the final concentration of SDS to 0.01%and of PBS to 0.01 molar. Allow the solution to incubate for 20 minutes. Next, increase the concentration of sodium chloride to 0.05 molar by adding a solution of two molar sodium chloride and 0.01 molar PBS.Maintain the SDS concentration at 0.01%and let the solution incubate for anoher 20 minutes. Increase the concentration of sodium chloride in increments of 0.1 molar over 20 minute intervals until a final concentration of one molar is reached. Incubate the solution at 23 degrees Celsius overnight on a rotator.After the DNA samples and gold nanoparticles have become conjugated, centrifuge the solution at 7, 000 times g for 30 seconds. Remove the supernatant. Next, prepare a 25 nanomolar gold nanoparticle probe solution by resuspending the pellet in deionized water.Prepare target DNA samples as outlined in the text protocol. Add six microliters of gold nanoparticle probe solution into each DNA sample with sodium chloride. Using a vortex mixer, shake each sample solution for five minutes at 23 degrees Celsius.Then, add six microliters of 400 millimolar hydroxyl amine and six microliters of 25.4 millimolar chloroauric acid to each DNA sample solution. Set these samples aside as a steady change in coloration will occur over the next hour. the size, shape-dependent optical properties of the nanoparticle, the number of the mismatch in a sample DNA fragment typical can be discerned by eye.To begin fabricating the droplet manipulation platform, utilize a computer numerical controlled machine equipped with a 0.5 millimeter drill bit to produce PMMA-based mastermolds with microstructures. For the drill, use a feed speed of seven millimeters per second and a rotation rate of 26, 000 rpm, and inspect the molds after drilling. Use an air blower and deionized water to clean the surface of the mastermolds and remove any PMMA scrap.Following this, prepare a 10 to one mixture of PDMS base and curing agent. Degas the mixture in a desiccator until all air bubbles are removed. Then, pour the PDMS mixture into the mastermold.Bake for three hours at 60 degrees Celsius to obtain the inverse microstructures in the air chambers and air channels. After baking is complete, remove the PDMS layer from the mastermold, and punch holes in it for the air suction inlets. Use an oxygen plasma treatment system to clean the PDMS membrane and PDMS layer.After treatment, bond the PDMS membrane and the PDMS layer, and then punch holes in this conjugated structure. Next, bond the PDMS and glass substrate together using a hot plate at 90 degrees Celsius for 10 minutes. Once the layers are bonded together, place the composite plate into a laser cutting machine PDMS membrane-side up.Import the dwg file to define the super hydrophobic area, and note this will be directly engraved into the PDMS membrane. Clean the newly engraved superhydrophobic surface with deionized water. This will wash away any carbonized residues.After the surface has been cleaned, connect the air suction inlet and vacuum pump through a solenoid valve. Then, connect the solenoid valve to a computer to control it with a digital IO USB module. Once the pneumatic droplet manipulation platform has been set up, add 10 microliters of 0.5 micromolar target DNA on the superhydrophobic area.Next, add 10 microliters of freshly hybridized 25 nanomolar gold nanoparticle probe solution next to the target DNA drop. Use a simple program to control air suction to the platform through a solenoid valve. Set the pressure to negative 80 kilopascals, and the driving frequency to five hertz.Provide suction along the droplet's vibration paths, causing them to collide and coalesce. Next, mix the coalesced droplet by manipulating the suction to roll the sample for three minutes. Add two microliters of 400 milimolar hydroxylamine, and two microliters of 25.4 milimolar chloroauric acid to the superhydrophobic area of the pneumatic droplet manipulation platform.Manipulate the suction so that the droplets collide and merge. Then, mix the merged droplet by rolling it for one minute. Observe the color of the final droplet with the naked eye.In this study, multi nucleotide polymorphisms are detected colorometrically using DNA hybridization mediated growth of gold nanoparticle probes. Here, the DNA samples possess different affinities to the probe DNA, causing differences in the growth of the gold nanoparticles, seen as a change in color. The three base pair mismatched DNA sample has less hybridization than the fully complimentary DNA, which can be seen as noticeable distinctions in color, especially at higher concentrations.The six base mismatched DNA has weak hybridization affinity to the probe, and thus the growth size of the gold nanoparticles was small. This colorometric analysis is shown to be repeatable when samples are manipulated via a pneumatic suction platform, as the results of DNA mismatch are readily observable by the naked eye. Once mastered, the entire procedure takes less than five minutes from the addition of the sample and the reagent until result of them, which is much quicker than the traditional method.The total in each operation is only 10 microliter, which is significantly less than what is required in a large system. Following this procedure, once the sequence variation of the space of DNA fragment is confirmed to be responsible for a specific disease. The specifically designed product can be used to acquire a number of mismatch of the sample DNA.The pneumatic droplet manipulation platform is more biocompatible than other methods such as optical weighting, dye electro fluorescence, electro weighting, and shoomoo's capillary actuation. The proposed multinucleotide polymorphism detection method was conducted under pneumatical droplet manipulation platform, which means the samples and reagents can be directly loaded and collected with pipette. After its development, this technique has a high potential for DNA screening, and other chemical and biomatical applications.

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Research

JoVE Journal

Genetics

7.5K ビュー.  National Taiwan University. The overall goal of this procedure is to combine a simple method for visually detecting multi-nucleotide polymorphisms with a pneumatic droplet manipulation platform. The DNA detection platform is on an open surface and is straight forward to use for biochemical researchers. We investigated a simple tool to repeatedly detect genetic mutations and genetic diseases.It has greater potential helping us understand how genetic variation leads to disease. We combined two novel and simple meth...