INTEGRATED MICROFLUIDIC SYSTEM for DETECTION of BIOMARKERS in BIOLOGICAL SAMPLES Hsiang-Yu Wang 1, Ming Yu 2, and Adam T

INTEGRATED MICROFLUIDIC SYSTEM FOR DETECTION OF BIOMARKERS IN BIOLOGICAL SAMPLES Hsiang-Yu Wang 1, Ming Yu 2, and Adam T. Woolley 2 1 Department of Chemical Engineering, National Cheng Kung University, Tainan, 701 Taiwan 2 Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA

ABSTRACT We have developed a polymer-based, integrated microfluidic system, which is capable of performing protein separation and fluorescence labeling in series, for the detection of targeted biomarkers inside cells or body fluids. The device incorporated a side channel to transport a fluorogenic dye, 3-(4-carboxybenzoyl) quinoline-2- carboxaldehyde) or CBQCA, to the end section of the separation channel for the post-column tagging of proteins of interest. We have successfully fabricated microchips from poly(methyl methacrylate), PMMA, and performed separation and labeling of model analytes as well as a widely used biomarker, alpha-fetoprotein (AFP), using these devices.

KEYWORDS: Biomarker detection, Post-column labeling, Microfluidics

INTRODUCTION The presence and levels of certain biomarkers in an individual serve as key indi- cators in the diagnosis of cancers, Alzheimer’s disease and many other illnesses. Traditional methods for detecting these biomolecules often involve multiple, complex, and laborious processes. Microfluidic systems for biomarker detection are ad- vantageous compared with conventional methods in several ways. First, integration of several processes into one device dramatically simplifies tasks and decreases the required amount of labor. Moreover, the tiny sample size needed and potential for high sensitivity in detecting biomolecules make microfluidic systems an ideal platform for trace molecule analysis. In this report, we demonstrate a microfluidic device capable of conducting sample separation and labeling in series. The biological samples used include myoglobin (MGB), AFP, and trypsin digested MGB. The results indicate that our devices were able to perform separations with better resolution than those using pre-column-labeled samples. We were also able to perform quantitative analysis of some samples using these microchips.

EXPERIMENTAL The polymer-based devices (Fig. 1) were fabricated from PMMA using hot em- bossing [1]. CBQCA was obtained from Molecular Probes (Eugene, OR, USA). The 488-nm line from an air-cooled Ar ion laser was used to excite samples, and emitted light (filtered via a 505LD dichroic) was collected using a photomultiplier tube.

Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences November 1 - 5, 2009, Jeju, Korea

Separation Channel 4 40 mm (25 mm) Figure 1. Schematic of our integrated microfluidic system. Fluid reservoirs: (1) Post-column Labeling sample, (2) buffer, (3) sample Detection (8 mm) waste, (4) labeling reagent, Point 5 and (5) waste.

RESULTS AND DISCUSSION We first performed the integrated assays using myoglobin (MGB), and resulting peak heights were linearly proportional to MGB concentrations in the range of 1-10 µg/ml. The lowest MGB concentration detected in these assays (10 mM KCN + 15 mM CBQCA) was 1 µg/ml (Fig. 2). Alpha-fetoprotein (AFP) is a commonly used biomarker for the detection of pregnancy, fetal health [2], and liver cancer [3]. In healthy adults, AFP levels are lower than 20 ng/ml, and variations in AFP amounts can be indicative of various health conditions [4]. We were able to detect AFP concentrations as low as 1 ng/ml using the integrated system (Fig. 3). Although the resulting peak heights did not increase linearly with AFP concentration, the variations in peak heights were sufficient to provide clinically meaningful information. The post-column labeling reduced peak broadening effects due to multiply labeled variants (Fig. 4), an advantage that is espe- cially notable during separation of a MGB digest (Fig. 5). The ability of our system to obtain sharp and well-separated peaks for complex protein mixtures shows its great potential for detection of biomarkers in body fluids.

CONCLUSIONS Conventional pre-column labeling processes often take more than 30 minutes, while our system only requires less than a minute to complete separation and labeling. Our system should improve throughput in biomarker detection, while reducing assay cost, labor and complexity. This rapid and effective platform should also provide benefits for clinical diagnosis, medical research, and other areas in life sciences requiring detection of targeted molecules.

ACKNOWLEDGEMENTS We thank the U.S. National Institutes of Health (EB006124) for supporting this work.

REFERENCES [1] R. T. Kelly and A. T. Woolley, Thermal Bonding of Polymeric Capillary Elec- trophoresis Microdevices in Water, Anal. Chem., 75, pp 1941–1945, (2003). [2] G. J. Mizejewski, Levels of alpha-fetoprotein during pregnancy and early in- fancy in normal and disease states, Obstet Gynecol Surv, 58, pp 804-825, (2003). [3] P. Alexander, Fetal antigens in cancer, Nature, 235, pp 137-140, (1972).

Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences November 1 - 5, 2009, Jeju, Korea

978-0-9798064-2-1/µTAS2009/$20ⓒ2009CBMS [4] M. Christiansen, et al., Alpha-fetoprotein in plasma and serum of healthy adults: Preanalytical, analytical and biological sources of variation and construction of age-dependent reference intervals, Scand J Clin Lab Inv, 61, pp 205-215, (2001).

MGB AFP * * 2.5 (µg/ml) (ng/ml) 1.5 2 10 1000 100 1.5 8 1 F.I. 6 F.I. 10 1 4 2 0.5 1 0.5 1 0 0 0 0 0 20 40 60 0 20 40 60 Time (s) Time (s)

Figure 2. Electropherograms (offset verti- Figure 3. Electropherograms (offset ver- cally) of post-column labeled MGB. Reac- tically) of post-column labeled AFP. tion conditions: 10 mM KCN and 15 mM Reaction conditions: 20 mM KCN and CBQCA (7.5% DMSO) in 10 mM sodium 20 mM CBQCA (10 % DMSO) in 10 borate buffer (pH 9.25) containing 0.5 % mM sodium borate buffer (pH 9.25) HPC. containing 0.5 % HPC

1.5 * 1.5

1 post-column 1 post-column F.I. F.I. 0.5 pre-column 0.5 pre-column 0 0 0 20 40 60 80 0 20 40 60 Time (s) Time (s) Figure 4. Electropherograms (offset verti- Figure 5. Electropherograms (offset ver- cally) of post-column and pre-column la- tically) of post-column and pre-column beled MGB (10 µg/ml). Reaction condi- labeled MGB digest, 8 µg/ml. Reaction tions are the same as in Figure 2. conditions: 10 mM KCN and 30 mM CBQCA (15% DMSO) in 10 mM sodium borate buffer (pH 9.25) containing 0.5 % HPC.

Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences November 1 - 5, 2009, Jeju, Korea