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 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.

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 probe DNA (100 µM, prepared in step 1.1) to a solution of gold nanoparticles (13 nm, 17 nM) at concentration 1 OD/ml.
   2. Bring the final concentrations of sodium dodecyl sulfate (NaC₁₂H₂₅SO₄, SDS) and phosphate buffer (PBS) to 0.01% and 0.01 M, respectively.
   3. After 20 min, bring the concentration of sodium chloride (NaCl) to 0.05 M with a solution of NaCl (2 M) and PBS (0.01 M) while maintaining SDS at 0.01% and incubate for 20 min.
   4. Increase the NaCl concentration in increments of 0.1 M to a final concentration of 1 M over 20 min intervals.
   5. Incubate overnight with shaking on a vortex mixer at 23 °C. The shaking speed does not affect the experiment as long as the DNA samples and AuNP probes become hybridized.
   6. Centrifuge the gold nanoparticles at 7,000 x g for 30 sec and remove the supernatant.
3. Add AuNP probe particles into deionized water (DI water) at a concentration of 25 nM (AuNP probe solution).
4. Prepare target DNA samples (fully complementary: 5'-CTTGCCTACGCCACCAGCTC-3'; three base-pair mismatched: 5'-CTTGCCTACTTTACCAGCTC-3'; six base-pair mismatched: 5'-CTTGCCTTTTTTTCCAGCTC-3') at concentrations of 0.06, 0.11, 0.17, 0.20, 0.30, 0.50 µM, respectively.
5. Add AuNP probe solution (6 µl, 25 nM) into the DNA samples (350 µl) with NaCl (14 µM).
6. Shake the mixture of DNA samples and AuNP probes with a vortex mixer at 23 °C for 5 min for DNA hybridization.
7. For AuNP growth, add NH₂OH (6 µl, 400 mM) and HAuCl₄ (6 µl, 25.4 mM) to the solution (the mixture of DNA samples and AuNP probes). The growth of AuNP takes approximately 30 sec for completion. The steady change of coloration maintains more than 1 hr.
   CAUTION: Skin contact with HAuCl₄ might produce severe toxic effects. Please consult the material safety data sheets (MSDS) before use. Please wear gloves and use a fume cupboard when using HAuCl₄.

2. Fabrication of a Pneumatic Droplet Manipulation Platform

Note: The PDMS-based droplet manipulation platform comprises two components: a PDMS membrane (100 µm) with a super-hydrophobic surface and an air-channel layer (5 mm). Without a MEMS process, the common machining processes were utilized to fabricate this device, which includes computer-numerical control (CNC) micromachining for making a mold, PDMS casting and replication for rapid prototyping of the microfluidic components, and laser micromachining for the fabrication of a super-hydrophobic surface (see Figure 3).

1. Use the CNC machine equipped with a drill bit (0.5 mm) to produce PMMA-based master molds (see Figure 3) with microstructures. Use a feed speed of the drill bit 7 mm/sec and a rate of rotation 26,000 rpm. Use an air blower and DI water to remove the PMMA scrap and to clean the surface of the master molds.
2. Prepare a mixture of the PDMS base and the curing agent in ratio 10:1. Degas the mixture inside a desiccator for 30 min, or until all air bubbles are removed.
3. Pour the PDMS into the master mold and bake the PDMS for 3 hr at 60 °C in the oven to obtain the inverse microstructures of the air chambers (PDMS membrane) and air channels (PDMS layer).
4. Remove the PDMS layer from the master mold (by hand). Punch holes on the PDMS layer for air-suction inlets.
5. Put the PDMS membrane, the PDMS layer and the glass substrate into the chamber of the oxygen-plasma treatment system. After oxygen-plasma treatment, bond the PDMS membrane, the PDMS layer and the glass substrate together on a hotplate (90 °C) for 10 min.
6. Put the composite chip into the laser-cutting machine (PDMS membrane side up). Import the .dwg file to define the superhydrophobic area (15 mm × 12 mm). See Figure 3. Directly engrave the PDMS membrane with a CO₂-laser machine to create the superhydrophobic area on the PDMS membrane (power 12 W, scanning velocity 106.68 mm/sec).
7. Clean the superhydrophobic surface with DI water to wash away the carbonized residues on the surface.
8. Connect the air-suction inlet and vacuum pump through a solenoid valve (see Figure 4). Connect the solenoid valve to the computer and control with a digital I/O USB module.

3. Operation for Detection of MNP on the Pneumatic Platform

Note: This section describes the operation to identify colorimetrically and rapidly the MNP on the pneumatic droplet manipulation platform (see Figure 5). All steps take place at 23 °C (ambient temperature) and relative humidity 85%.

1. Place the target DNA sample droplet (10 µl, 0.5 µM) on the superhydrophobic area of the pneumatic droplet manipulation platform.
2. Place the AuNP probe droplet (10 µl, 25 nM) on the superhydrophobic area of the pneumatic droplet manipulation platform.
3. Control the air suction to cause the deflection of the PDMS membrane with a solenoid valve and a simple program (e.g., Labview). Provide air suction in each air chamber along the migration path of the droplets (containing target DNA and AuNP probes respectively) on the PDMS membrane such that the droplets collide with each other and coalesce. Use a working pressure of the vacuum pump of -80 kPa (gauge pressure).
   1. Alternatively, control the air suction manually as long as the droplet is fully mixed. By hand, connect or disconnect the vacuum pump and inlets of air chamber with tubes in the early stages of testing.
4. Control the air suction to roll the coalesced droplet forward and backward to enhance the mixing efficiency for 3 min using the controlling system of the solenoid valve. The target DNA and AuNP probes become well mixed and hybridized within 3 min. Use a driving frequency and a working pressure of 5 Hz and -80 kPa (gauge pressure), respectively.
5. Place a droplet containing NH₂OH (2 µl, 400 mM) and HAuCl₄ (2 µl, 25.4 mM) on the superhydrophobic area of the pneumatic droplet manipulation platform.
6. Control the air suction (-80 kPa gauge pressure) to let the droplets collide with each other and coalesce. Then control the air suction (5 Hz and -80 kPa gauge pressure) to let the coalesced droplet roll forward and backward to enhance the mixing efficiency for 1 min.
7. Measure and record the color of the coalesced droplet with the naked eye.

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