PROTOTYPE MICRO GAS CHROMATOGRAPH FOR BREATH BIOMARKERS OF RESPIRATORY DISEASE

S. K. Kim1, H. Chang1, and E .T. Zellers1,2*

Center for Wireless Integrated Microsystems (WIMS), Departments of 1Environmental Health Sciences, 2Chemistry, University of Michigan, Ann Arbor, MI, USA

ABSTRACT Progress toward a prototype MEMS gas

chromatograph (μGC) designed for analyzing complex mixtures of volatile organic compounds (VOC) in breath is described. This µGC, named SPIRON, integrates a multi-stage preconcentrator/injector, a dual-microcolumn separation module, and a chemiresistor array detector with commercial minivalves and a minipump. Here, we describe results showing thermal control of all key system components and then present the rapid separation of two complex mixtures: one containing lung cancer biomarkers and the other containing tuberculosis biomarkers, both in backgrounds of 30 common breath VOCs. The fully assembled SPIRON µGC prototype is also presented. KEYWORDS

Lung cancer, tuberculosis, breath biomarker, μGC, micro column, sensor INTRODUCTION

It has long been known that the analysis of breath samples can be used as a non-invasive diagnostic tool for certain diseases [1-3]. Many investigations have focused on lung cancer, because it is the leading cause of cancer death in the U. S. and the five-year survival rate can be 60-80% if the disease is diagnosed at an early stage [4]. Another major lung disease is tuberculosis (TB), which is the foremost cause of death from a single infectious agent in the world, causing more than 26% of avoidable adult deaths in the developing countries [5].

Simple methods for early non-invasive diagnosis of lung cancer, TB and other diseases are needed. The instrument most commonly used for analysis of volatile organic compounds (VOC) in breath is a gas chromatograph coupled with a mass spectrometer detector (GC-MS) because of its versatility for determining a wide range of volatile analytes [6]. Efforts to miniaturize MS and GC-MS systems have been mounted by several groups and organizations [7-10].

In this research, we are developing a micro gas chromatograph (μGC), referred to as SPIRON, and

tailoring its design and operating parameters to the problem of determining biomarkers for lung cancer and TB in breath. The challenge is to differentiate the biomarkers from endogenous and exogenous VOC interferences at low- or sub-parts-per-billion concentrations in a matrix nearly saturated with water vapor. The layout diagram of the analytical subsystem of the SPIRON µGC is shown in Figure 1. It is based on a previous prototype µGC developed in our group [11]. The key components of the instrument are a microfabricated 3-stage preconcentrator/focuser (µPCF), two 3-meter-long DRIE-Si/glass microcolumns with a non-polar PDMS stationary phase coated on the interior walls, and a thermo-electrically cooled chemiresistor (CR) array that uses thiolate-monolayer-protected gold nanoparticle (MPNs) films as the interface layers. Commercial valves are used as well as a commercial miniature rotary-vane pump. The microcolumns are independently temperature-programmable from room temperature to ~200 ºC. The system also uses heated MEMS interconnects. Scrubbed air is used as the carrier gas to avoid the need of cylinder gas. Based on published research and our own GC-MS measurements of breath from healthy subjects and indoor air samples collected from 5 different sites, 7 biomarkers of lung cancer, 6 biomarkers of tuberculosis [1-3], and 30 common interferences have been selected for initial studies. The lung cancer markers are isoprene, pentane, 2-methylpentane, ethylpropanoate, benzene, toluene, and ethylbenzene [1,2]. The markers for pulmonary tuberculosis are 1,4-dimethylcyclohexane, 3-heptanone, 2,2,4,6,6-pentamethylheptane, p-cymene, and 1-methylnaphthalene [3]. The work described here is focused primarily on component-level testing. High-speed separations of lung cancer and TB biomarkers from potential interferences are shown using a conventional flame ionization detector. The use of integrated heaters and software-based controllers for rapid μPCF and microcolumn heating is illustrated, along with rapid changes and in the operating temperature of the

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sensor array using a TE cooler. The assembled SPIRON µGC prototype is also presented.

Figure 1. Fluidic layout diagram of the SPIRON system RESULTS AND DISCUSSION Thermal Control of Microsystem Components As shown schematically in Figure 2, a control program written in Labview (National Instruments, Austin, TX) was used with a 16-bit DAQ card to operate all the components and to process the signal readouts from the sensor array. A pulse-width-modulated (PWM) square wave function generated by the DAQ is used to control the rate of heating or cooling for all components. The active time of PWM square wave is calculated by proportional-integral-derivative (PID) algorithm [12].

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Figures 5 and 6 show the separations of the lung cancer and TB biomarkers from 30 common breath VOC constituents, respectively. For these tests the dual-column ensemble was mounted in the oven of a bench-scale GC, connected to the injector and FID detector, and it was temperature programmed from room temperature to 150°C. The 7 biomarkers of lung cancer are separated in 4 min, even though the remaining compounds require 7.5 min to elute. The 6 TB biomarkers are separated in ~ 8 min. Air was used as a carrier gas. Figure 7 shows response patterns from a CR array for 5 of the 7 lung cancer biomarkers obtained in separate testing (unpublished) with a portable GC using the CR-array detector [13]. Unique response patterns and detection limits in the low-ppb range are achieved for most of the biomarkers by preconcentrating a 1-L sample volume. However, early-eluting biomarker peaks are obscured by the relatively large residual-water peaks even though a short dry-air purge of the preconcentrator was incorporated into the analytical protocol. The chromatogram shown in the lower portion of Figure 7 is from the C8-MPN sensor, which is the least water sensitive sensor. Other sensors in the array give larger water vapor peaks. Use of Nafion tubing at the inlet succeeded in removing a significant fraction of water vapor from the sample, but additional measures are needed to completely resolve this problem.

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Figure 5. Temperature-programmed dual-3-m-microcolumn chromatogram of 7 lung cancer breath biomarkers (boxes) and 30 common breath-VOC interferences.

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Figure 7. Normalized response patterns for 5 lung cancer biomarkers obtained by the CR-sensor array from separate tests in a portable GC. The acronyms in the response patterns refer to the structures of the ligands of the MPNs: n-ctanethiolate (C8), 1-mercapto-6-phenoxyhexane (OPH), 7-mercaptoheptanitrile (CCN), and methyl 6-mercaptohxanoate (HME) and the name of compounds: 2-methylpentane (2MPEN), benzene (BZ), ethylpropanoate (EPRO), toluene (TOL), and ethylbenzene (EBZ). Single-sensor (C8) chromatogram trace of lung cancer biomarkers and 30 common VOCs found in normal human breath. System Integration The assembled SPIRON µGC, consisting of a printed-circuit board populated with all components, is shown in Figure 8. The PC board dimensions are 10 cm × 12 cm. In order to capture quantitatively VOCs spanning a wide range in vapor pressure and to thermally desorb them efficiently for separation, as well as to reduce water vapor uptake, the 3-stage µPCF is packed with two hydrophobic graphitized-carbon adsorbents Carbopack B (1st stage: 4270 × 2785 × 380 μm) and Carbopack X (2nd stage: 2405 × 2780 × 380 μm), and a hydrophobic carbon molecular sieve Carboxen 1018 (3rd stage: 1615 × 2779 × 380 μm). The two 3-m-long DRIE-Si/glass microcolumns have cross-sections of 150 × 240 µm and a 0.15 µm thick wall-coated PDMS stationary phase. The columns have on-board heaters and RTDs for temperature programming of

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the separations. The MPN-coated CR array is mounted on the TE cooler assembly through a cut-out in the PC board. The system also uses heated MEMS interconnects which accommodate fused-silica capillaries to reduce possible adsorptive losses to surfaces of the transfer lines. Commercial valves are used as well as a commercial mini-pump to direct flow. A control program in Labview is being written to be used with a PC-installed 16-bit DAQ card to operate all the components and to process the signals from CR array.

Figure 8. The SPIRON µGC. ACKNOWLEDGMENTS

The authors wish to thank Katharine Beach for microcolumn fabrication, Brendan Casey for wire bonding, Robert Gordenker for PC board design and system integration, and Dr. Qiongyan Zhong for the data in Figure 7. This work was supported by the National Science Foundation Engineering Research Centers Program under Award ERC-9986866, by a Pilot Project Research Grant from the National Institute for Occupational Safety and Health, and by a grant from Intel Corporation.

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CONTACT * E.T. Zellers, tel: +1-734-936-0766; ezellers@umich.edu

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