HIGH SENSITIVITY PROTEIN DETECTION USING MICRO-MAGNETIC APTAMER PCR (MAP) TECHNOLOGY Aren E. Gerdon1, Jiangrong Qian1, Yanting Zhang2, Jonathan D. Adams1, Seung Soo Oh1, Andrew Csordas1, and H. Tom Soh1 1University of California, Santa Barbara, USA, and 2Cynvenio, LLC,USA

ABSTRACT Direct detection of protein biomarkers from complex mixtures over a wide range of concentrations is a significant challenge for clinical biosensor technology. In this work, we present Micro-magnetic Aptamer PCR (MAP) biosensor technology for specific, quantitative protein detection at pico-molar concentrations, directly from blood serum. The biosensor system integrates three complimentary technologies: a micro-magnetic separation chip to purify target proteins from complex mixtures, a DNA aptamer affinity reagent that binds to the target protein and interfaces with PCR amplification, and real-time PCR to quantitate protein concentrations. As a model system we demonstrate the detection of Platelet-Derived Growth Factor (PDGF), a cancer biomarker, in undiluted serum over four orders of magnitude from 1 nM to a detection limit of 124 + 6 fM.

KEYWORDS: PCR, Protein Detection, Magnetic, Aptamer

INTRODUCTION The Micro-magnetic Aptamer PCR (MAP) biosensor system allows specific, quantitative protein detection at pico-molar concentrations, directly from blood se- rum (Fig 1).

Figure 1. The MAP protein detection technology; (A) magnetic beads presenting the capture-antibody and PCR-aptamer are added to a complex mixture containing the target protein; (B) antibody/target/aptamer complex is formed; (C) non-specific proteins as well as unbound aptamers are removed via micro-magnetic separation; (D) the antibody/target/aptamer is eluted in pure buffer; (E) PCR amplification and quantitative detection of the target is achieved via real-time PCR.

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA

978-0-9798064-1-4/µTAS2008/$20©2008CBMS 1594 Compared to conventional protein sensors, such as the Enzyme-linked Immu- nosorbant Assay (ELISA),[1] and other PCR amplification-based sensors, such as Immuno-PCR,[2] the MAP sensor provides significant advantages. First, specificity is achieved through dual protein recognition by two affinity reagents (polyclonal an- tibodies and DNA aptamers) for capture and signaling (Fig 1 A, B). The use of ap- tamers to integrate with PCR, as opposed to antibodies commonly used in Immuno- PCR, obviates the need for tedious bioconjugation. Only the protein targets recog- nized by both the antibody (on magnetic beads) and the PCR aptamer (in solution) will form an antibody/target/aptamer complex and generate signal. Second, the tar- get complex is purified via a micro-magnetic separation chip (MMS) to discard the background contaminants which interfere with the subsequent PCR amplification (Fig 1 C, D). Third, we achieve amplification and quantification of the analyte via real-time PCR (Fig 1 E). In this way, we obtain quantitative data that is directly pro- portional to the concentration of target protein in the serum sample.

RESULTS AND DISCUSSION As a model target, we chose platelet-derived growth factor (PDGF), a protein cancer marker involved in tumor angiogenesis[3,4] that is present at serum concen- trations as low as 1 pM.[5,6] Here, we demonstrate hundred femtomolar detection of PDGF in undiluted blood serum with a dynamic range over four orders of magni- tude. DNA aptamers and antibodies that recognize the B subunit of PDGF were used to bind PDGF-BB, allowing successful protein capture and signaling (Fig 2).[8] The an- tibodies and aptamers used in the experiment were highly specific and did not bind to the PDGF-AB heterodimer or PDGF-AA homodimer (data not shown).

Figure 2. Aptamer structure determined by mFold[7] calculations. (a) Aptamer se- quence, determined from literature,[8] matches well with the structure previously reported; (b) Addition of 20 nucleotide PCR primer binding sites at the 5’ and 3’ ends does not disrupt aptamer fold.

The MMS chip, fabricated on glass substrates with integrated ferromagnetic struc- tures (Ni), was used to efficiently trap the magnetic beads and target sandwich com- plexes. The background contaminants from serum were removed by washing with buffer for 5 minutes at 20 ml/hr, and the target sandwich complexes were eluted in buffer for real-time PCR detection. The limit of detection (LOD) for the PDGF protein was 124 + 6 fM in undiluted fetal bovine serum and the detection range extended over

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 1595 four orders of magnitude (Fig 3). This LOD is approximately 1,600x lower than those obtained via Biacore SPR (LOD = 0.2 nM) using the same affinity reagents, and dem- onstrates the advantages of the MAP biosensor system for quantitative detection of rare protein markers from complex mixtures.

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20 PCR Threshold Cycle, C Cycle, Threshold PCR

15 1.0E-18 1.0E-15 1.0E-12 1.0E-09 1.0E-06 [PDGF-BB], M Figure 3. Detection of PDGF-BB in undiluted fetal bovine serum. The sensor showed concentration dependence over four orders of magnitude and the limit of de- tection was 124 + 6 fM at a signal to noise ratio of 3:1.

REFERENCES [1] Crowther, J.R., The ELISA Guidebook. (Humana Press, Totowa, New Jersey, 2001). [2] Niemeyer, C.M.; Adler, M., and Wacker, R. "Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification." Trends Biotech. 23, 208 (2005). [3] Yu, J.; Ustach, C., and Kim, H-R. C. "Platelet-derived Growth Factor Signaling and Human Cancer." J. Biochem. Mol. Biol. 36, 49 (2003). [4] Ross, R.; Raines, E.W., and Bowen-Pope, D.F. "The Biology of Platelet- Derived Growth Factor." Cell 46, 155 (1986). [5] Zhang, B-B.; Cai, W-M.; Weng, H-L.; Hu, Z-R.; Lu, J.; Zheng, M., and Liu, R- H. "Diagnostic value of platelet derived growth factor-BB, transforming growth factor-B1, matrix metalloproteinase-1, and tissue inhibitor of matrix metalloproteinase-1 in serum and peripheral blood mononuclear cells for he- patic fibrosis." World J Gastroenterol 9, 2490 (2003). [6] Czarkowska-Paczek, B.; Bartlomiejczyk, I., and Przybylski, J. "The serum lev- els of growth factors: PDGF, TGF-Beta and VEGF are increased after strenu- ous physical exercise." J. Phys. Pharm. 57, 189 (2006). [7] Zuker, M. "Mfold web server for nucleic acid folding and hybridization predic- tion." Nuc. Acids Res. 31, 3406 (2003). [8] Green, L.S.; Jellinek, D.; Jenison, R.; Ostman, A.; Heldin, C-H., and Janjic, N. "Inhibitory DNA Ligands to Platelet-Derived Growth Factor B-Chain." Bio- chemistry 35, 14413 (1996).

Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 1596