Biosensor Array Strategies for Cancer Biomarker Proteins

1. Biosensor Array Strategies for Cancer Biomarker Proteins James F. Rusling Professor of Chemistry and Cell Biology University of Connecticut and University Health Center Collaboration with F. Papadimitrakopoulos (IMS) and Drs. Gutkind and Patel (NIH, Bethesda) Website: http://web2.uconn.edu/rusling/ Email: James.Rusling@uconn.edu

2. Cancer Biomarkers: National Institutes of Health – molecules that can be objectively measured and evaluated as indicators of normal or disease processes and pharmacologic responses to therapeutic intervention Cancer Biomarker Proteins: increase in serum concentration At the onset of cancer, even before tumor develops • Multiple proteins must be measured for reliable predictions • Excellent hope for early detection and treatment monitoring • May also be able to monitor inflammation • May facilitate new therapies

3. Key aspects and needs: • Ultrasensitive detection of multiple cancer biomarkers • Long term objectives • 1. early cancer detection and monitoring • 2. tools for cancer research and surgical decisions • • point-of-care (POC) clinical assays – need to be cheap, simple, fast, accurate, multiplexed • Expensive, and complex methodologies such as LC-MS/MS, some automated optical-based methods are currently not competitive for POC Reviews:

4. Biomarker Targets: 1. Prostate Specific Antigen • PSA - Single chain glycoprotein , MW 33 kDa Sensitive, specific biomarker for detection of prostate cancer years before clinical signs of disease • Detection of PSA in serum: clinical detection of prostate cancer: 4-10 ng/mL • Led to less invasive treatment protocols, avoid surgery Adapted From Brookhaven Protein Databank • 2. Interleukin 6 (IL-6) • - prostate and oral cancer biomarker • - human plasma conc. normal < 6 pg/mL; cancer 20-1000 pg/mL

5. ELISA- enzyme linked immuno-sorbent assay 96-well plate Antigen = Protein, pathogen Well in plate Secondary antibody and labels enzyme label Primary antibody Antibodies capture The antigen Detection by optical absorbance plate reader after running enzyme reaction that gives a colored product • Reliable method for over 30 years • Best DL ~ 3 pg/mL in serum • many commercial assay kits for single proteins • limitations in sample size, speed, multiplexing

6. Possible approaches: • fluorescence labels • surface plasmon resonance, SPR • Electrochemiluminescence (ECL) –Ru(bpy)32+ labels • bead based assays, ECL or Fluorescence S Slope = sensitivity Detection limit = blank signal + 3x avg. noise Conc. protein

7. Multilabel Strategies – high sensitivity • detection by fluorescence, amperometry, ECL • non-specific binding must be minimized in any immunoassay

8. Possible multilabel strategies Dissolve, measure M+n Also used on particles Up to 400,000 labels

9. SPR arrays – measure refractive index at interface – potentially label free -More susceptible to Non-specific binding (NSB)

10. Bead-based protein assays + Label – enzyme; Fluorophor; DNA, RuBPY(ECL) Protein in sample multi-enzyme- Magnetic bead-Ab2 Multiple labels Bead captures protein, Magnetic separation, wash Detection: • ECL – RuBPY label • Fluorescence – Fl. Label • 1-10 pg/mL DL • $200,000 for machine Magnet under well in 96-well plate, wash to remove NSB

11. 2. 1. A conventional Single electrode AuNP film Multilabel magnetic particle, on-line capture B Electrochemical Immunosensors Capture Antibody Ab2-enzyme Multi-enzyme- Magnetic particle-Ab2 AuNP Protein analyte

12. AuNP-based immunosensors – single sensors nanostructures sensor + massive multilabel strategies Antibodies on AuNPs AFM, carboxylated-AuNPs on polycation underlayer Rotating disk amperometry (A) and calibration for PSA on AuNP platform AuNPs: DL for PSA = 0.5 pg/mL (8 x)

13. Accurate PSA detection in cell lysates and patient serum Using AuNP-based immunosensors

14. SWNT array for 4 prostate cancer proteins in human serum vs. ELISA 8-sensor array Bhaskara V. Chikkaveeraiah,Ashwin Bhirde, Ruchika Malhotra, Vyomesh Patel, J. Silvio Gutkind, and James F. Rusling, Single-Wall Carbon Nanotube Forest Immunoarrays For electrochemical measurement of 4 Protein Biomarkers for Prostate Cancer, Anal. Chem., 2009, 81, 9129–9134.

15. 5 nm Au nanoparticle vs. single wall nanotube electrodes • head to head comparison on IL-6 detection show better Detection limits and better linearity with AuNPs • AuNPs are easier to handle and produce highly reproducible electrodes Bernard S. Munge, Colleen E. Krause, Ruchika Malhotra,Vyomesh Patel, J. Silvio Gutkind, and James F. Rusling, Electrochemical Immunosensors for Interleukin-6. Comparison of Carbon Nanotube Forest and Gold Nanoparticle platforms, Electrochem. Comm., 2009, 11, 1009–1012

16. Off-line capture magnetic particle microfluidic strategy A Conventional, single label array electrode Multilabel magnetic Particle, off line capture B Electrical contacts -0.2V + H2O2 + HQ  signal 8 electrodes In channel Capture Antibody Ab2-enzyme 1 mm multi-enzyme- Magnetic particle-Ab2 7000-400,000 labels AuNP Protein analyte

17. 8-electrode PDMS microfluidic array electrodes Electrical contacts

18. Components of microfluidic device made of micro-machined polymethylmethacrylate), soft PDMS microfluidic channel + screen printed 8 electrode carbon array

19. Microfluidic protein assay system

20. Microfluidic array detection of PSA in serum samples Using off-line capture with 1 m multilabel magnetic particle; Detection limit ~100 fg/mL PSA in 10 L serum; 5-fold better than manual assay without off-line capture

21. Microfluidic array data for mixture of PSA and IL-6 in serum Flow rate : 100 µL/min H2O2 : 100 µM Hydroquinone : 1 mM Limit of detection of PSA: 225 fg/mL Limit of detection of IL6 : 300 fg/mL

22. Ab1 PSA IL6 PSA 1-3% IL6 2-5% Low cross reactivity of PSA and IL-6

23. Immunosensor assay validation on human serum PSA IL-6

24. Serum Protein Biomarkers for Oral Cancer • Interleukin-6 [IL-6] • Interleukin-8 [IL-8] • Vascular Endothelial Growth Factor [VEGF] • Vascular Endothelial Growth Factor C [VEGF-C] Trikha, M.; Corringham, R.; Klein, B.; Rossi, J. Clin. Cancer Res. 2003, 9, 4653-4665 Hebert, C. A.; Baker, J. B. Cancer Invest. 1993, 11, 743-750 O-charoenrat, O.; Rhys-Evans, P.; Eccles, S. A. Cancer 2001, 92, 556-568

25. Microfluidic Immunoarray: Oral Cancer Biomarkers Off-line capture using magnetic particles with 400,000 HRPs IL-6 DL: 10 fg mL-1 IL-8 DL: 15 fg mL-1 VEGF-C DL: 60 fg mL-1 VEGF DL: 8 fg mL-1

26. Protein Array using RuBPY ECL label Immunoassays in 10 mL wells Carbon (PG) chip (no microelectronics) SWCNT forest ECL = Electrochemiluminescence Detection labels are 100 nm d. silica with internal RuBPY

27. Forster and Voss, 1980s (synthesis) RuPVP Solid PG chip Spots contain capture antibody on RuPVP ECL polymer

28. ECL arrays for detection of PSA and IL-6 DL ~ 0.1 pg/mL 2 ng/mL 0.2 ng/mL 0.1 pg/mL 0 Control

29. Surface plasmon resonance (SPR) detection of protein biomarkers using superparamagnetic beads labels

30. SPR response to PSA in Serum with Magnetic and Silica labels Using off-line capture with 1 m magnetic particle label; Detection limit ~10 fg/mL PSA

31. Ultrasensitive multiple protein arrays: • Combining nanostructured sensors or SPR with (multi-label) magnetic particles gives ultrahigh sensitivity in fg/mL range • Microfluidics with off-line analyte protein capture gives very low S/N, semiautomated • May open door to new ultralow abundance biomarkers • ECL provides simpler array for protein detection, no microelectronic chip needed

32. Cancer Biomaker Protein Measurements • as yet, limited POC or clinical use except for PSA • ELISA, commercial kits, one protein, 3 pg/mL DL • bead based methods, up to 10 proteins, equip. and kits expensive, 1-10 pg/mL DLs • LC-MS, great for discovery, emerging for routine tests • new experimental methods promise ultrasensitivity, detection in fg/mL range, multiplexing - microfluidic amperometric arrays, multilabel - ECL arrays, simplicity - SPR arrays with magnetic particle labels - fiber optic microwell arrays – D. Walt (Tufts) - DNA label “bar-codes” - C. Mirkin (Northwestern)