NON-CONTACT HEAT MANAGEMENT FOR NUCLEIC ACID HYBRIDIZATION IN SAMPLE-TO-ANSWER CENTRIFUGAL MICROFLUIDICS
R. Gorkin*1,3, K. Abi-Samra1,3, F. Begin2, Gale Stewart2, M. Bergeron2, H. Kido1, and M. Madou1,3
1
Department of Biomedical Engineering, University of California - Irvine, CA, USA
Centre de Recherché en Infectiologie de l’Université Laval - Québec, City, Québec, Canada
3
World Class University (WCU), Ulsan National Institute of Science and Technology, Ulsan, South Korea
2
ABSTRACT
This report focuses on the application of a newly developed non-contact heat management system, based on infrared
radiation, to achieve advanced nucleic acid (NA) hybridization on a centrifugal microfluidic platform. Our system was
used to eliminate formamide, a common reagent used to enable hybridization at room temperatures. Formamide poses
significant challenges in the development of molecular µTAS platforms with respect to issues such as reagent storage
and low analyte detection. Thermal energy can be used to replace formamide during NA hybridization, allowing for the
processing of more targets and the elimination of a highly corrosive reagent. The developed heat management system
was implemented to produce highly stringent conditions for formamide-free NA hybridization of genetic material from
clinical samples containing parainfluenza viruses on a rotating microfluidic platform.
KEYWORDS: Centrifugal - Compact Disc - CD Microfluidics, Nucleic Acid, Hybridization
INTRODUCTION
Heating is essential for virtually all steps in NA assay processing. For example, heating is necessary for proper elimination of PCR inhibitors from raw samples, for accurate and rapid thermocycling during PCR, and for correct nucleic
acid hybridization in microarray detection. As centrifugal microfluidics continues to aim to develop a sample-to-answer
diagnostic platform capable of nucleic acid detection [1], heat management will take an increasingly important role in the
development of the field. To help meet this goal, we report on the development of a non-contact heat management system that addresses the challenging task of tight temperature control for accurate NA hybridization for pathogen identification in an integrated microfluidic device.
THEORY
In terms of nucleic acid hybridization, molecular biologists use the term “stringency” to describe the conditions of establishing the non-covalent and sequence specific interactions between complimentary strands. Specifically, stringency
refers to the ability of single stranded NA molecules to discriminate between complementary and non-complementary
sequences during hybridization. Additionally, stringency refers to the measure of the likelihood that duplexes of NAs
will dissociate into single strands [2,3]. Thus, the higher the stringency, the more likely stable duplexes will form; similarly, the lower the stringency the less likely stable duplexes will form as annealing occurs with some degree of mismatching.
Temperature plays an important role in determining the degree of stringency. At critical temperatures, thermal energy will break the hydrogen bonds holding two NA strands together. These temperatures are generally just above the
melting point (Tm) where 50% of double stranded NAs are dissociated into their constituent single strands, and high
stringency hybridization takes place just below the Tm. At these precise temperatures, only the individual NA strands
with exact matching sequences will have the greatest chance to anneal [2,3].
In order to perform hybridization in native laboratory conditions and to prevent damage to genetic material by overheating, organic solvents, particularly formamide, are added to create high stringency conditions at room temperatures.
Formamide works by forming hydrogen bonds with the base pairs in DNA and replacing the native ones. This results in
less stable duplexes which can be more easily dissociated, shifting the Tm of the DNA to a lower temperature. However,
in terms of sample-to-answer molecular analysis the use of formamide is particularly problematic. The use of formamide,
being a solvent that cannot be dried, presents problems with storage; as dried reagents are ideal for manufacturing, transport, handling and use in clinical settings. Additionally, large fractions of formamide (~30%) often need to be added to
buffers in order to bring down the Tm to room temperatures. In many diagnostic assays based on clinical samples, the hybridization solutions consist of low sample volumes with low analyte concentrations. By replacing formamide with more
sample, one can process samples with higher concentrations, allowing for more accurate pathogen detection. Thus, for a
µTAS system for nucleic acid detection, it would be advantageous to eliminate formamide entirely.
EXPERIMENTAL
The experimental setup consisted of a heating platform built around a motor spin-stand to drive the centrifugal microfluidics and disposable cartridges for microarray hybridization (Figure 1). For temperature regulation, infrared (IR) heating and thermometry elements were linked and controlled via automated feedback. A halogen lamp, encased in a reflector receptacle (International Light Technologies, USA), and a compact infrared sensor (Optris, Germany) were
positioned above the rotor. The IR sensor was coupled to a data acquisition board connected to a desktop computer. The
computer was also connected to a programmable power supply (Instek, Taipei, Taiwan) which powered the IR lamp. The
complete system was controlled by Labview (National Instruments, USA) where a customized program was written to
control the thermal output via a power supply and acquire temperature readings via the IR sensor.
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
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14th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
3 - 7 October 2010, Groningen, The Netherlands
Figure 1: A) In-house fabricated IR heating housing unit on portable motor unit – shown in open position. B) Unit in
closed position. C) Picture of heating during rotation.
The microarray cartridges consisted of 1) a PDMS part, containing reagent storage reservoirs and the connecting
microfluidics, and 2) a glass slide pre-spotted with the microarray (target specific capture probes) (Figure 2). For assembly, the PDMS part was then overlaid and aligned to a glass slide with a microarray. Before experimentation, the raw
samples were prepared by culturing several viral strains of parainfluenza (PIV) followed by the homogenization of the
culture to isolate the genetic material. Reverse transcription PCR (RT-PCR) was performed in order create the amplified
target sequences needed for detection (i.e., RT-PCR is necessary to create double stranded genetic material as PIV is
RNA based). After processing, the amplicons were enzymatically dissociated to create ssDNA for hybridization. Once
complete, the reservoirs of the hybridization cartridge were loaded. The sample reservoir was loaded with 2 µL of hybridization mix (8X SSPE, 0.4% PVP and post-PCR material), the wash reservoir was loaded with 10 µL of wash buffer
(0.2X SSPE + 0.1% SDS), and the rinse reservoir was loaded with 10 µL of rinse buffer (0.2X SSPE).
Figure 2: Microfluidic hybridization cartridges (PDMS molds on printed array slides) are placed in the bottom rotor
recesses and secured. The pictures illustrate the sequential emptying of the sample, wash and rinse reservoirs during increased accelerations.
Loaded cartridges were then placed on a rotor and the system was secured (Figure 2). When the preparation was complete, a set spin protocol was started. The protocol followed four discrete accelerations; one to break each of the valves
restricting the hybridization mix, wash and rinse buffers respectively, and a final spin to remove residue fluid from the
chambers. Immediately after the motor engaged, the IR heating program was initiated. After the completion of the first
acceleration, the heat was removed (as only the hybridization requires heat). Following the full spin program, the
cartridges were removed from their housings. Then PDMS parts were removed and the slides examined with a microarray scanner (Agilent DNA Microarray Scanner). Images taken from the scanner were then processed with analyzing
software (GenePix Pro 6.1) to collect fluorescence intensities.
RESULTS AND DISCUSSION
To identify the appropriate conditions for formamide-free hybridization, experiments were performed to narrow the window of temperatures that resulted in equivalent signal intensities to formamide based mixes. The three sets of bar graphs on
the right in Figure 3 represent the target sample, three strains of cultured PIV (the label ending in H26a being the most
prominent, while H26b, and H22a are slight mutated forms). The set of bars on the very left in Figure 3 represent a
hybridization control and ensured that annealing took place. In terms of temperature, the bar with 23°C (room temperature with no added heat) shows the solution with formamide. The remaining bars represent formamide-free hybridization mixes under varied heated conditions. Two types of hybridization solutions were examined. One replaced formamide
with an equivalent amount of buffer, while the second replaced formamide with extra amplicons. The goal was to attain signals as intense or greater with either of the formamide-free solutions (requiring the raised temperature).
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Figure 3: Hybridization Results. By heating formamide-free samples at different temperatures (30°C-36°C) equivalent signal intensities could be seen compared to samples with formamide (23°C). Replacing the buffer with extra amplicon produced the highest fluorescence signals. (The left-most of the four set of bars shows hybridization control)
The results of the experiments helped to narrow the range of appropriate working temperatures from 30°C-36°C. For
the first two strains of PIV tested, the results closely mirrored each other. At 30°C, 33°C, and 36°C the formamide-free
solutions with extra buffer showed signal intensity similar to the original formamide solution at 23°C. In addition, in the
cases where formamide was replaced with extra amplicons, as compared to the formamide-free solutions at the same
temperature, equivalent or higher signal intensities were observed. The increased signal intensity from the added amplicons can be attributed to the increased number of fluorescence producing targets (i.e., more source, more signal). With
the third PIV strain, some signal was seen with the formamide-free solutions (only trace fluorescence was seen with the
formamide buffers); however, no correlation can be seen. Taken as a whole, the system is able to produce almost equivalent fluorescence with the same amount of targets without formamide, and by increasing the temperature of the system.
While this ability is by no means trivial, in terms of producing the highest fluorescent signal, the optimal conditions are
to replace the formamide with amplicons.
In terms of a NA µTAS CD-based system, the heating platform showed the ability to perform biological assays at
elevated temperatures. For NA hybridization, an essential component of the current CD design, the removal of formamide has significant benefits. First, it allows for the inclusion of more target to create an improve signal intensity for a
given sample. Second, it solves the potential engineering difficulties that arise when using a corrosive non-dryable solvent.
CONCLUSION
Our report shows a novel contact-free heating technique for use on a rotating microfluidic platform, validated by performing an advanced NA hybridization assay. The ability to integrate controlled heating with centrifugal microfluidics
opens up the possibility for next generation biological and chemical analysis on a disc. In the immediate future, the platform can be used for testing the various assay steps requiring heating previously outlined as essential in NA analysis.
While this system has been proven for hybridization, the use for more demanding processes like PCR would test the system in the most extreme conditions utilized in µTAS NA analysis and warrant further study.
REFERENCES
[1] R. Gorkin, J. Park, J. Siegrist, M. Amasia, B.S. Lee, J.M. Park, J. Kim, H. Kim, M. Madou, and Y.K. Cho, Centrifugal microfluidics for biomedical applications. Lab on a Chip, 10, 1758-1773, (2010)
[2]
Farrell, R. RNA methodologies – A laboratory guide for isolation and characterization 3rd edition, Elsevier, Inc.,
(2005)
[3] Roche Applied Science, Switzerland. (2009) DIG Application manual for in situ hybridization – Chapter 3 Nucleic
acid hybridization – general aspects. Available www.roche-applied-science.com
CONTACT
*R.A. Gorkin III, tel: +1-949-8875496; [email protected]
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