NON-CONTACT HEAT MANAGEMENT FOR NUCLEIC ACID HYBRIDI-ZATION 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 1Department of Biomedical Engineering, University of California - Irvine, CA, USA

2Centre 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

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 elimi-nation 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 sys-tem that addresses the challenging task of tight temperature control for accurate NA hybridization for pathogen identifi-cation in an integrated microfluidic device.

THEORY

In terms of nucleic acid hybridization, molecular biologists use the term “stringency” to describe the conditions of es-tablishing 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; simi-larly, the lower the stringency the less likely stable duplexes will form as annealing occurs with some degree of mis-matching.

Temperature plays an important role in determining the degree of stringency. At critical temperatures, thermal en-ergy 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 over-heating, 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, trans-port, 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 hy-bridization 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 micro-fluidics and disposable cartridges for microarray hybridization (Figure 1). For temperature regulation, infrared (IR) heat-ing and thermometry elements were linked and controlled via automated feedback. A halogen lamp, encased in a reflec-tor 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 115 14th International Conference onMiniaturized 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 assem-bly, 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 hy-bridization 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 in-creased accelerations.

Loaded cartridges were then placed on a rotor and the system was secured (Figure 2). When the preparation was com-

plete, 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 microar-ray 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 win-dow 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 tem-perature with no added heat) shows the solution with formamide. The remaining bars represent formamide-free hybridiza-tion 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 sig-nals 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) equiva-lent signal intensities could be seen compared to samples with formamide (23°C). Replacing the buffer with extra ampli-

con 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 ampli-cons 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 equiva-lent 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 forma-mide 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 sol-vent. CONCLUSION

Our report shows a novel contact-free heating technique for use on a rotating microfluidic platform, validated by per-forming 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 plat-form 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 sys-tem 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, Cen-

trifugal 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; robertgorkiniii@gmail.com

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