Attomolar Detection of a Cancer Biomarker Protein in Serum by Surface Plasmon Resonance Using Superparamagnetic Particle Labels

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1 Attomolar Detection of a Cancer Biomarker Protein in Serum by Surface Plasmon Resonance Using Superparamagnetic Particle Labels Challa V. Kumar, 1,3 Vigneshwaran Mani, 1 Sadagopan Krishnan, 1 Dhanuka Wasalathanthri, 1 Amit Joshi,1 James F. Rusling 1,2 1 Department of Chemistry, University of Connecticut, Storrs, CT 2 Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 3 Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT Ultrahigh sensitive immunosensor 1

2 Introduction Protein Biomarkers are elevated levels of proteins in blood serum, indicative of the onset, existence or progression of cancer. 1 Accurate measurement of panel of proteins in biological fluids holds enormous potential for directed personalized therapy and treatment monitoring. 1 For example, early detection of protein biomarkers such as PSA, IL-6 holds great promise in multiple cancer diagnosis, prognosis and disease monitoring. 1. Rusling J.F., Kumar C.V., Gilkind J. S., Patel V, Measurement of 2 biomarker proteins for point of care early detection and monitoring of cancer, Analyst, 2010, 135, PSA- Prostate specific antigen, IL-6 interleukin-6

Contd. Protein biomarkers Normal levels Early stage cancer levels PSA 0.

3 Contd. Protein biomarkers Normal levels Early stage cancer levels PSA ng ml -1 4 to 10 ng ml -1 (prostate) IL-6 < 6 pg ml -1 > 20 to 1000 pg ml -1 (Oral) Serum levels of IL-6 are 1000 fold smaller than those of PSA (in pg/ml levels) Further, reoccurrence of prostate cancer after post radical prostatectomy (surgical removal of prostate) leads to PSA levels in sub fg/ml-pg/ml. There are very few methods which can detect such a low levels of protein biomarkers 3 1. Ruth Etzioni, et al. Nature reviews, volume 3, Shad Thaxton, PNAS, vol 106, 2009

4 Current methods Enzyme-linked immunosorbent assays (ELISA) DL ~1 pgml -1. (Expertise required) Commercial kits such xmap technology by Luminex, Roche s ELECSYS (ECL), Mesoscale multiarrays, bead based assays can measure 500 or more proteins. (DL~1-10 pg/ml) However, expensive $200000/machine and $ per assay. Only, centralized labs and research institution can afford

5 Need for point of care, inexpensive strategies to measure ultralow levels of protein biomarkers in serum at sub pg/ml levels amidst thousands of irrelevant sera proteins. We achieved by Surface plasmon resonance coupled with offline capture of proteins using one micron magnetic beads to detect ultralow levels of protein biomarkers 5

6 Surface plasmon resonance (SPR) Surface sensitive optical technique, monitors biomolecular interactions in real time and label free. Provides specificity (yes or no binding response), concentration analysis, kinetics (rate of reactions), affinity analysis of (strength of binding) Current applications study protein- protein interactions, ligand receptor binding, DNA hybridization, protein folding, drug/ inhibitors interaction to proteins 6

SPR Phenomenon Kretschmann configuration Evanescent wave(~200nm) I

i Incident light Resonance occurs θi> θ c Reflected light θ c θ

Energy(E) and momentum(m) incident photons = E and M of surface

transferred to evanescent wave leading to decreased intensity at

7 SPR Phenomenon Kretschmann configuration Evanescent wave(~200nm) I i 50 nm glass e - e - e - Au film e - e - e - Prism θ i I r I r /I i Incident light Resonance occurs θi> θ c Reflected light θ c θ SPR Angle of Incidence Material with free electrons Au,Ag Energy(E) and momentum(m) incident photons = E and M of surface plasmons At resonance, a part of incident light energy is transferred to evanescent wave leading to decreased intensity at the reflected light. 7 Evanescent wave: Standing wave, decays exponentially with distance

$refractive index change at the interface.$

8 How SPR works? 50 nm glass e - e - e - Au film e - e - e - Prism I i θ i I r I r /I i Incident light θi> θ c Reflected light θ c θ SPR Angle of Incidence Antibody Protein SPR is very sensitive to refractive index change at the interface. µriu k a k d Change in the angle is converted to resonance signal which is directly proportional to adsorbed mass 8 Time, sec

Conventional SPR immunosensor Conventional Way Fluidic flow Ø Low sensitivity is an issue Capture antibody Light Source Prism Detector Biomarker

9 Conventional SPR immunosensor Conventional Way Fluidic flow Ø Low sensitivity is an issue Capture antibody Light Source Prism Detector Biomarker protein Our strategy (Offline capture of biomarker) SPR signal ampli5ication Super paramagnetic Particle Labels MP + MP 9 Dynabeads Detection antibody

10 Offline Capture Detection antibody coated MP(MP-Ab2) + Minimizes non- speci5ic binding PSA MP-Ab2-PSA Offline capturing PSA MP facilitate sample handling MP-Ab2-PSA Signi.icantly high SPR response was detected compared to the control Control 1µm silica particles (SP) Light Source Prism Detector Capture antibody(ab1) 10

11 Why magnetic particles Advantages Commercial available magnetic particles with different functional groups 11 Facilitate sensitive methodologies Available from 100 nm to 50 µm Paramagnetic beads useful for protein capture, separation and transport Signal amplifying agents in SPR Inexpensive, technically undemanding, automating protein detection

12 Primary antibody (ligand) immobilization chemistry Mixed self-assembled monolayer on Gold PEG to resist non specific binding Active group to immobilize biomolecules 12 Reichert

13 Magnetic particle-antibody conjugate Binding Chemistry Superparamagnetic Tosyl 1 µm MP Tosyl Activated Magnetic Particle 1) Anti-PSA Secondary Antibody (24 hr) 2) BSA Blocking(16 hr) MP MP-Ab 2 Conjugate Proteins Magnetic Particle-Protein conjugate Dynabeads MyOne Tosylactivated 13 MP Prostate Specific Antigen (PSA) in Calf Serum (90 min) MP-Ab 2 -PSA Conjugate in PBS-T Analyte

14 Real time curve for anti-psa Immobilization (Ligand) Activation with EDC/NHS Blocking with Ethanolamine Anti-PSA in 10mM NaOAc ph 5.5 2% BSA 14 EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide NHS:N-Hydroxysuccinimide

15 A. SPR sensograms of PSA- MP- Ab 2 Conjugates binding to covalently Immobilized Ab 1 on sensor surface B. In.luence of PSA concentration on maximum SPR signals Ultra low detection limit - 10 fg/ml ( 300 am) in calf serum Krishnan, 15 S.; Mani, V.; Wasalathanthri, D.; Kumar, C. V.; Rusling, J. F. Angewandte Chemie, 2011,

16 Scanning electron microscopy images magnetic MP Ab2 PSA (5 pgml - 1 PSA) nonmagnetic control SP Ab2 PSA(5 pgml - 1 PSA) bound to Ab1 on gold SPR surfaces 16

17 Dynamic Light Scattering MP-Ab 2 SiP-Ab 2 A) MP-Ab 2 -PSA at No PSA B) SiP-Ab 2 -PSA at No PSA MP-Ab 2 SiP-Ab 2 A) MP-Ab 2 -PSA at 5pg/mL PSA B) SiP-Ab 2 -PSA at 5 pg/ml PSA 17 Magnetic particles clustering is independent of PSA concentration in solution, and No clustering for SiPs

18 Patient serum samples Sample 4 control sample Samples 1,2,3 were from patient with prostate cancer 18 SPR immunosensor results for patient serum samples compared with standard ELISA Samples were diluted fold in PBS-T

19 Interleukin-6 19 SPR sensograms of IL- 6- MP- Ab 2 Conjugates binding to covalently Immobilized Ab 1 (1800uRIU) on sensor surface Ultra low detection limit - 10 fg/ml ( 300 am) in diluted calf serum B. In.luence of IL- 6 concentration on maximum SPR signals

20 Amplification of SPR signal Magnetic particles acts as intrinsic labels in SPR Ø High refractive index of magnetic particles Ø Aggregation of particles on the gold surface (SEM) Ø Superparamagnetic plasmonic coupling 20

21 Objectives: ü To investigate the antibody-antigen binding characteristics of antibody-magnetic particle (Ab 2 MP) conjugates K = 21 D k k d a k d k a Why it is important? k d k a k a = Association constant k d = Dissociation constant K D = Equilibrium dissociation constant = Antigen = secondary antibody = Magnetic bead To identify relevant binding epitopes for immunosensor application. Why SPR? ü Real time analysis ü Label free technique

transport Mass transport Unstirred layer

22 Antigen Antibody binding kinetics Theoritical point of view Flow Flow Mass transport Mass transport Unstirred layer 1. Higher flow rate (> 30 µl /min) low mass transport effect 2. Low surface ligand densities (< 130 µru) minimize the rebinding of dissociated analytes 22 Myszka,D.G.; Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors; Curr.Opin. Biotechnol.; 1997; 50-57

A + B k a AB Analyte ligand k d Analyte ligand complex k d k a Association [A]/C = Equilibrium concentration of A [B] = Equilibrium concentration of B [B 0 ] = Initial

23 A + B k a AB Analyte ligand k d Analyte ligand complex k d k a Association [A]/C = Equilibrium concentration of A [B] = Equilibrium concentration of B [B 0 ] = Initial concentration of B [AB] = Equilibrium concentration of AB At t>0, conc. of unoccupied ligand, [B] = [B] o - [AB] SPR response (R) α [AB] Max. Response (R max ) = [B 0 ]

24 After integration of equation 3 4 Dissociation d[ab]/dt = -k d [AB] d[r]/dt = -k d [R] 5 After integration of equation R t = R e 0 k d t 6 24 K = D k k d a SPR response / mru injection Association Dissociation Time /s

25 Curve fitting Graphpad prism software Utilizes Non-linear regression Association Model Based on equation 4 Dissociation Model Based on equation 6 25 Bmax is the maximal binding at equilibrium, at maximal radioligand concentration.

26 Results Anti-PSA Ab 2 binding PSA Association Kinetics Dissociation Kinetics 26 k a = 7.63± 0.16 * 10 4 k d = 8.93± 0.09 * 10-4 K d = 11.7 nm Association time = 300 s Dissociation time=1hr Flow rate= 50 µl/min Extended Dissociation Curve fitting using GraphPad Prism Software

27 Anti-IL-6 Ab 2 binding IL-6 antigen Association Kinetics Dissociation Kinetics k a =4.14 ± 0.17 *10 4 k d =9.2 ± 0.3 *10-4 K d = 22.2 nm 27 Association time = 300 s Dissociation time=1hr Flow rate= 50 µl/min Extended Dissociation One phase exponential decay Curve fitting using GraphPad Prism Software

28 MP-Ab 2 binding PSA antigen 28 Association time = 360 s Dissociation time=360s Flow rate= 30 µl/min Running Buffer: 20 mm PBS 150mM NaCl % tween + 0.1% BSA Double Referenced Curve fitting was not possible either Scrubber/graphpad prism

29 MP-Ab 2 binding to IL-6 29 Association time = 360 s Dissociation time=360s Flow rate= 30 µl/min Running Buffer: 20 mm PBS Curve fitting was 150mM NaCl % tween + 0.1% BSA not possible either Double Referenced Scrubber/graphpad prism

30 30 Rate constant for MP-Ab 2 binding IL-6, PSA using initial slope analysis

31 High ligand density µriu PSA IL-6 31 Ligand Density(Ab1): 1000 uriu Rate Constant: 2.2 * 10 7 Ligand Density(Ab1): 1000 uriu Rate Constant: 2.2 * 10 7

32 Low ligand density µriu PSA IL-6 Ligand density(ab1) : 100 uriu Rate constant: 8.8 * 10 6 Ligand density(ab1) : 300 uriu Rate constant: 1.5*

33 MP-Ab2 binding PSA (low and high density) Association rate constant High Density (1000 uriu) Low Density (100 uriu) Rate constant : 2.2 * 10 7 Rate constant : 8.8 * 10 6 Dissociation rate constant No dissociation Ab2 binding PSA k a = 7.63± 0.16 * 10 4 k d = 8.93± 0.09 * 10-4 K d = 11.7 nm MP-Ab2(high and low density) k a >>>>>k a of Ab2 binding PSA MP-Ab2 dissociation kd <<<<<<<kd of Ab2 binding PSA MP-Ab2 KD <<<<<Ab2 binding PSA 33

34 MP-Ab2 binding IL-6 (low and high density) Association rate constant High Density (1000 uriu) Low Density (300 uriu) Rate constant : 2.2 * 10 7 Rate constant : 1.5 * 10 7 Dissociation rate constant No dissociation Ab2 binding IL-6 k a =4.14 ± 0.17 *10 4 k d =9.2 ± 0.3 *10-4 K d = 22.2 nm MP-Ab2(high and low density) k a >>>>>k a of Ab2 binding IL-6 MP-Ab2 dissociation kd <<<<<<<kd of Ab2 binding IL-6 MP-Ab2 KD <<<<<Ab2 binding IL-6 34

35 Future work Multiplexed detection of cancer biomarkers using Surface plasmon resonance imager PSA Ab1 IL-6 Ab1 PSA-MP-Ab2 MP PSA MP MP IL-6 35 MP IL-6-MP-Ab2

36 Summary q We demonstrated an unprecedented low DL for cancer biomarker protein at am levels in serum using SPR immunosensor with MP amplification q This approach should also be applicable to other proteins and small molecules q Results show excellent correlation with ELISA over clinically relevant range of PSA. q SPR kinetics study reveal highly efficient capture of biomarker proteins using magnetic particles bioconjugated with antibodies. 36

37 Acknowledgements NIH for funding 37

Surface Plasmon Resonance for Immunoassays. Sadagopan Krishnan Chem 395 Instructor: Prof.Rusling

Surface Plasmon Resonance for Immunoassays Sadagopan Krishnan Chem 395 Instructor: Prof.Rusling 1 Outline Introduction Understanding the Basics SPR- Instrumental components Applications in Immunoassays