Name: the visual colorimetric detection of multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform
Uploaded: 2016-09-27T14:00:00
Description: Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

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.

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			<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. ...

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