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/http://web2.uconn.edu/rusling/ Email: James.Rusling@uconn.eduJames.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. enzyme label Primary antibody 96-well plate Antigen = Protein, pathogen Secondary antibody and labels ELISA- enzyme linked immuno-sorbent assay Antibodies capture The antigen Well in plate 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) 3 2+ labels bead based assays, ECL or Fluorescence S Conc. protein Slope = sensitivity Detection limit = blank signal + 3x avg. noise

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

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

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

10. multi-enzyme- Magnetic bead-Ab 2 Multiple labels + Protein in sample Bead captures protein, Magnetic separation, wash Magnet under well in 96-well plate, wash to remove NSB Detection: ECL – RuBPY label Fluorescence – Fl. Label 1-10 pg/mL DL $200,000 for machine Bead-based protein assays Label – enzyme; Fluorophor; DNA, RuBPY(ECL)

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

12. AuNP-based immunosensors – single sensors nanostructures sensor + massive multilabel strategies AFM, carboxylated-AuNPs on polycation underlayer Antibodies on AuNPs 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 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. 8-sensor array

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. Ab 2 -enzyme 1  m multi-enzyme- Magnetic particle-Ab 2 7000-400,000 labels AuNP Capture Antibody A B array electrode Electrical contacts 8 electrodes In channel Protein analyte Conventional, single label Multilabel magnetic Particle, off line capture Off-line capture magnetic particle microfluidic strategy -0.2V + H 2 O 2 + HQ  signal

17. Electrical contacts electrodes 8-electrode PDMS microfluidic array

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 H 2 O 2 : 100 µM Hydroquinone : 1 mM Limit of detection of PSA: 225 fg/mL Limit of detection of IL6 : 300 fg/mL

22. Low cross reactivity of PSA and IL-6 2-5%IL6 1-3%PSA IL6PSAAb 1

23. Immunosensor assay validation on human serum PSAIL-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 IL-6 DL: 10 fg mL -1 IL-8 DL: 15 fg mL -1 VEGF DL: 8 fg mL -1 VEGF-C DL: 60 fg mL -1 Off-line capture using magnetic particles with 400,000 HRPs

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

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

28. Control 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

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)