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

Summary

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 °C without the aid of advanced instruments.

Abstract

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 µl in each operation, which is remarkably less than that of a macro system.

Introduction

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 hig....

Protocol

1. Method to Detect MNP

Note: This section describes the procedure to detect the MNP based on the hybridization-mediated growth of gold nanoparticles.

1. Prepare the probe DNA (5'-thiol-GAGCTGGTGGCGTAGGCAAG-3') solution at a concentration 100 µM.
2. 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 be conducted. We typically prepare excess AuNP probe particles as spares. The volume used in these steps is adjustable and flexible.
   1. Add pr....

Results

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........

Discussion

In this protocol, a simple colorimetric method to detect MNP can be implemented at concentrations ranging from 0.11-0.50 µM 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PDMS	Dow Corning	SYLGARD 184
benchtop engravers	Roland DG	EGX-400
laser cutting machine	Universal Laser Systems, Inc.	VLS 3.50
Oxygen plasma treatment system	Femto Science Inc. Korea	CUTE-MPR
solenoid valve			home built
vacuum pump	ULVAC KIKO, Inc.	DA-30D
13-nm AuNP solution	TAN Bead Inc., Taiwan	NG-13
DNA (with 5'-end labeled thiol)	MDBio, Inc., Taiwan
phosphate buffered saline (PBS)	UniRegion Bio-Tech,. Taiwan	PBS001-1L
sodium dodecyl sulfate (SDS)	J. T. baker	4095-04
Hydroxylamine solution (NH2OH)	Sigma-Aldrich	467804
Chloroauric acid (HAuCl4)	Sigma-Aldrich	G4022
sodium chloride (NaCl)
vortex mixer	Digisystem Laboratory Instruments Inc.	VM-2000
centrifuge	Hermle Labortechnik GmbH.	Z 216 MK