A PROGRAMMABLE MICROFLUIDIC SYSTEM FOR SELECTIVE RNA OR DNA EXTRACTION FROM VARIOUS RAW BIOLOGICAL SAMPLES Michael Johnson 1* , Jungkyu Kim 1, Angela Williams 2, and Bruce Gale 1 1University of Utah, UNITED STATES OF AMERICA and 2Integrated Explorations, CANADA ABSTRACT A system is presented that promises to be a step towards universal sample preparation in its ability to selectively extract and purify nucleic acids from various sources. A three-layer polydimethylsiloxane (PDMS) microfluidic chip is fabricated to perform the main fluid handling tasks. Nucleic acid is purified on the chip using standard solid phase extraction on a disposable glass fiber filter. The ability of the system to handle different sample inputs is demonstrated by extracting DNA from both a stock DNA solution and whole blood, and by extracting RNA form a both a stock RNA solution and living E. Coli cells. KEYWORDS: Nucleic Acid, Sample Preparation, Microfluidics INTRODUCTION Microfluidic systems are progressing towards the goal of reducing complex and time consuming laboratory analysis processes onto a single lab-on-a-chip device. These devices often rely on nucleic acid as the main analysis target because of its ubiquity, stability, and specificity. While genetic on-chip analysis techniques show great promise in expanding the role of microfluidic systems towards reducing human intervention in performing bio-assays, there is still a problem in that these sensing platforms demand purified nucleic acids, requiring typically manual genetic extraction and purification methods to remove unwanted biological material from the sample. This hurdle has limited the use of lab-on-a-chip devices to speciallized laboratories with the capability to perform the sample preparation steps, so widespread development of benchtop microfliudic analysis systems with true sample-in to answer-out capabilities is highly contingent on the development of nucleic acid purification sample preparation devices. Thus, while the number and type of microfluidic analysis techniques has grown in recent years, the practical use of lab-on-a-chip systems to perform wide- ranging laboratory-type tests has been limited and would be greatly benefited by the development of a sample preparation device that purifies genetic material from raw biological samples 1. The varied conditions of the sources of such material and the stringent requirements of analysis techniques force system-specific sample preparation operations, which often require time consuming and expensive manual handling 2. It would be of great benefit to have a self-contained lab-on-a- chip system capable of preparing genetic material out of raw biological samples from a variety of sources for microfluidic analysis. We present a system that promises to be a step towards universal sample preparation in its ability to take samples from various sources and selectively extract and purify nucleic acids (DNA or RNA). THEORY The main purpose of the sample preparation device is to extract and purify nucleic acids from biological samples. The nucleic acid is purified on the chip using standard solid phase extraction on a disposable glass fiber filter 3. The extraction process operates by pumping the sample and various reagents through integrated pipetting/mixing reservoirs and through an extraction filter where the nucleic acid is selectively bound and released depending on buffer conditions. Altering the amounts, types, and sequences of reagents used allows selective adjustment of the protocol to handle different sample types and control the output as DNA, RNA or both and is easily accomplished by adjusting the control program. The vision is to produce an entire extraction system, such as that seen in Figure 1, that provides a compact, portable control platform with minimal inputs. (electrical power, a USB connector, and an optional pressure supply). The polydimethylsiloxane (PDMS) microfluidic chip seen in Figure 2(a) that performs the main fluid handling tasks of the system is fabricated in three layers: the fluidic channel layer, Figure 1: Programmable Microfluidic Nucleic Aci d the functional flexible membrane layer, and the pneumatic Extraction System. This compact device uses control layer. The PDMS chip contains on-chip valves, electric vacuum and pressure pumps along with a channels, reservoirs, and pumps that are all pneumatically bank of solenoid valves to control a PDMS controlled via electrically actuated solenoid valves (Lee microfluidic chip. A computer connection allows Company). A LabView computer program (National easy programming to adjust the protocol to handle Instruments) operates the solenoid valves in sequence to a variety of sample inputs. 978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 387 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands Figure 2: PDMS Chip Overview. (a) Photograph of the fabricated microfluidic chip measuring 125 mm x 100 mm. (b) Diagram of the chambers and channels of the microfluidic chip with inputs labeled for extraction of RNA from E. coli cells. Input reagents can be easily adjusted for other samples. perform the required range of tasks such as sample input, lysis, incubation, mixing, extraction, cleaning, etc. The PDMS chip also has input ports for the reagents required to extract nucleic acid from a variety of samples. Figure 2(b) is a schematic of the main chip functional layers with labels indicating which reagents are connected to the input ports. Following extraction, the purified genetic material can be mixed with other reagents required for analysis techniques— electrochemical detection in this case. EXPERIMENTAL The automated system (Figure 1) contains a PDMS microfluidic platform complete with on-chip valves, reservoir pumps, and a disposable extraction filter. Fabrication of the PDMS chip involves a modified xurographic 4 technique for multi-level structures. Molds were created using a scotchcal vinyl tape (3M) cut on a sign plotter (Graphtec), with higher structures being cut from acrylic on a CO 2 laser and positioned over the tape with an adhesive. The control and operational fluidic layers are separated by a flexible silicone membrane (BISCO), and the layers are bonded using a novel masked corona discharge method. The disposable glass fiber filter used for solid phase extraction was fabricated from thin acrylic sheets and glass filter paper (Whatman). The acrylic sheets were cut on a laser and were bonded to enclose the glass filter with a double-sided adhesive. Known concentrations of RNA from an RNA standard stock (Invitrogen) were used as the sample. An RNA- containing solution was placed at the sample inlet port of the PDMS device. A protocol for on-chip mixing of the sample with reagents was used to bind the RNA to the glass filter, wash off any contaminants, and subsequently release the RNA in an elution of nuclease-free water. RNA extraction results were determined using a fluorescent Ribogreen RNA quantification kit obtained from Invitrogen. Fluorescence data was obtained using a 96- well plate reader (Bio-TEK). Binding of the nucleic acid to the PDMS and silicone surfaces was minimized using various coatings and solution stabilizers. RNA recovery yields from a sodium poly-phosphate (NaPP) coated system approached those from the commercial spin kit. However, these high-yield products displayed some NaPP contamination that interfered with reverse-transcript Figure 1: Manifold Mounting System. Photograph of polymerase chain reaction (RT-PCR) analysis methods. A the manifold mounting system that allows for rapid polyvinylperilidone (PVP) coating was used to produce replacement of the PDMS microfluidic chip. RT-PCR compatible outputs at the cost of a slightly reduced extraction yield. Nucleic acid extraction from raw biological samples was demonstrated by performing lysis and RNA extraction from E. Coli bacteria cells (New England Biolabs). The cells were incubated in cell culture medium and growth was monitored using optical density measurements to ensure viability. The E. Coli cells were then loaded into the automated system where they were enzymatically lysed, and RNA was extracted and quantified. Similarly, pre-purified human genomic DNA was used as the sample to be extracted in the system. The sample was drawn into the device and mixed with the appropriate reagents. The DNA extraction was quantified and verified by performing PCR on the eluate. Following verification of the ability to recover DNA, whole blood from a volunteer was used as the extraction sample and DNA was again extracted in the system and quantified using fluorescence and PCR. 388 A cleaning step was included in the program to allow for multiple tests on a single PDMS chip. A solution of RBS Neutral (Sigma-Adrich) is flushed twice over the system followed by three flushes of nuclease-free water (Qiagen). The programmable nature of the device allows for other disinfectants and cleaning agents to be introduced into the PDMS device, and the entire PDMS chip is also manifold-mounted [Figure 3] for easy replacement for sensitive tests where disposable units are desired. RESULTS AND DISCUSSION The device was used to demonstrate successful automated RNA recovery at yields approaching results attained by a commercial spin kit. The ability of the system to handle different sample inputs is demonstrated by extracting DNA from both a stock DNA solution and whole blood, and by extracting RNA form a both a stock RNA solution and living