Multiplexing immunoassay with SERS Neelam Kumarswami 24th Feb 29 5 4 3 42_421_44 42_421_47 42_421_47(2) 42_44_47 44_47_42(2) 421_44_47 421_44_47(2) Intensity 2 1 6 8 1 12 14 16 18
Aim of the project Robust, rapid, low cost, quantitative, multiplexed Point of Care (POC) biosensor platform for early diagnosis of human disease.
Monday, 2 March 29 Outline SERS particles for multiplexing and SERS technology Antibody-SERS conjugation Bioactivity of conjugates Raman data 3
What are SERS particle Inner gold core surrounded by SERS reporter molecules coated with glass. Average diameter 6-9 nm. Can used in a LFA format to quantitate multiple cardiac biomarkers within a single blood sample. Highly multiplexable Near IR detection Particles can be dried Uniform in size 4
SERS Technology Monday, 2 March 29 SERS is a surface technique arising from the enhancement of Raman scattering through molecules adsorbed on metal surfaces. Raman scattering itself is a result of an incident photon interacting with the electric dipole of a molecule resulting in inelastic light scattering. The energy from the vibration modes from SERS depends on the molecule s structure and environment Enhancement factor can be up to 1 14-1 15 Intensity is directly proportional to concentration of species 125.7546 663nm 785nm 851.1514 177.24 1231.666 1299.574 1617.5856 plasmons υ 6 7 8 9 1 11 12 13 14 R hif ( 1) 5
Monday, 2 March 29 Advantages of SERS Technology 1. Excellent sensitivity due to metal enhancement of the Raman signal. 2. Multiplexed capability through the use of various dyes to produce tags with unique spectra. 3. Ability to detect and quantitate in complex matrices, for example blood. 4. Fast detection system 5. Hand-held instrument 6. Compatible with existing lateral flow device manufacture and calibration 6
SERS selected for Multiplexing Monday, 2 March 29 D D D D N N N N D D D D SERS 42 SERS 421 N N N N SERS 44 SERS 47 Preliminary set of SERS particles selected based on better peak separation and resolution from Oxonica data 7
Raman spectra for selected SERS Monday, 2 March 29 851.1514 125.7546 177.24 1231.666 1299.574 25 2 15 1617.5856 1 4 42 35 Intensity 672.4323 736.1213 857.4 925.6719 13.7667 123.39 1 5 3 25 2 15 421 1282.1353 1428.2517 Intensity 1586.677 N N D D D D N N D D D D 5 6 7 8 9 1 11 12 13 14 15 16 17 18 6 7 8 9 1 11 12 13 14 15 16 17 18 45 4 35 3 25 2 721.1739 1213.684 825.476 12.235 1159.414 15 1284.6265 166.6459 Intensity 1 5 47 2 15 1 795.6943 851.1514 166.3726 1339.5 Intensity 1617.5856 1643.961 125.6462 126.984 N N 5 N 44 N 6 7 8 9 1 11 12 13 14 15 16 17 18 6 7 8 9 1 11 12 13 14 15 16 17 18 8
Monday, 2 March 29 Overlay of SERS spectra 4 35 3 Regions of variations 42 421 44 47 42 421 44 47 25 Intensity 2 15 1 5 6 8 1 12 14 16 18 R am an shift(cm -1) SERS 42 and 44 chosen for model system studies 9
Model System Monday, 2 March 29 Cardiac biomarkers Troponin I and Myoglobin For Troponin I: 1+2 antibody combinations investigated using 19C7-42 conjugates, with a mixture of Clones MF4 and 56 capture antibodies on membrane or ELISA plates For Myoglobin: 7C3 44 conjugates were prepared with 4E2 as the capture antibody. Control line Test Line 1
SERS-Antibody Conjugation Monday, 2 March 29 Mechanism Step 1: Attachment of protein onto a linker using NHS chemistry Step 2: Coupling of linker onto SERS particles Step 3: Quenching with MESA (2-mercaptoethanesulphonic Acid) MAb NH 2 NaO 3 S O incubation N O O N O Step 1 purification NH O N O O O O Sulfo-SMCC linker O S Step 2 SERS SH NH N O 3-4 hr O SERS-conjugate 11
Optimisation of conjugation minimum aggregation and best activity Monday, 2 March 29 Parameters Investigated: - Buffer type (Borate, Phosphate, Acetate, PBS) - Buffer ph (6.6, 7.2, 7.5,8) - Antibody loading (19C7/7C3) (9-3 μg/ml) Methodology: - Trial conjugations with 19C7 (tested visually) - Test conjugations with 19C7 (tested by ELISA) - Wet tests (lateral flow assays) - Raman Spectroscopy Final Selected Conditions: - 5mM Borate: ph 7.2 (Troponin); ph 6.6 (Myoglobin) - 3-5 μg/ml antibody loading 12
Characterisation of Conjugates Monday, 2 March 29 Nanosight Dynamic light scattering (DLS) ELISA (activity) Lateral flow assays (activity) Raman UV-Vis 13
Analysis by DLS of SERS-Conjugates (1) Monday, 2 March 29 Borate buffer ph 7.2 (2 days) Sodium acetate buffer ph 6.6 (2 days) 15 Size Distribution by Intensity 1 Size Distribution by Intensity Intens ity (% ) 1 5 Intens ity (% ) 8 6 4 2.1 1 1 1 1 1 Size (d.nm).1 1 1 1 1 1 Size (d.nm) Record 2: 42 in sodium borate 1 Record 3: 42 in sodium borate 2 Size Distribution by Volume Record 5: 42 IN NAOH 1 Size Distribution Record 6: by 42 Volume IN NAOH 2 Record 7: 42 IN NAOH 3 15 15 Volume (% ) 1 5 Volume (% ) 1 5.1 1 1 1 1 1 Size (d.nm).1 1 1 1 1 1 Size (d.nm) Record 2: 42 in sodium borate 1 Record 3: 42 in sodium borate 2 Record 5: 42 IN NAOH 1 Record 6: 42 IN NAOH 2 Record 7: 42 IN NAOH 3 14
Analysis by DLS of SERS-Conjugates (2) Monday, 2 March 29 PBS buffer ph 7.5 (2 days) Borate buffer - 1 week and 1 month Intens ity (% ) Size Distribution by Intensity 12 1 8 6 4 2.1 1 1 1 1 1 Size (d.nm) In te n s ity (% ) Size Distribution by Intensity 2 15 1 5.1 1 1 1 1 1 Size (d.nm) 15 Record 8: 42 IN PBS 1 Record 9: 42 IN PBS 2 Record 1: 42 IN PBS 3 Size Distribution by Volume Record 2: 42 in sodium borate 1 Record 3: 42 in sodium borate 2 Record 4: 42 in sodium borate 3 Record 17: 44 in sodium borate 7.2 ph 1 Record 18: 44 in sodium borate 7.2 ph 2 Record 19: 44 in sodium borate 7.2 ph 3 Volume (% ) 1 5.1 1 1 1 1 1 Size (d.nm) Minimum aggregation observed for borate buffer after 1 month Record 8: 42 IN PBS 1 Record 9: 42 IN PBS 2 Record 1: 42 IN PBS 3 15
Lateral Flow Assay(1) Analytes SERS conjugate Sample (blood, serum) Conjugate pad Test lines Control line Antibody Antibody Sample prefilter pad Nitrocellulose membrane 3-plexing Absorbent pad Liquid flows along path of least resistance
Lateral Flow Assay(2) Liquid flows along path of least resistance Analytes SERS conjugate Antibody Antibody Sample prefilter pad Sample (urine, serum) Conjugate pad Test lines Control line Nitrocellulose membrane 3-plexing x 3 conjugate pad Absorbent pad
Raman Mesophotonics Data(1) Monday, 2 March 29 Spectra from SERS manually deposited onto nitrocellulose Overlay of spectra for SERS 42, 44 and their conjugates 24 22 2 18 42 44 (42+44)conjugates 16 Intensity 14 12 1 42 42 44 8 6 4 2 Laser wavelength 663 nm 8 1 12 14 16 18 18
Raman Mesophotonics Data(2) 2 (42+44)conjugates 42+44 conjugates(1/1) 42+44 conjugates (1/6) 15 Intensity 1 5 8 1 12 14 16 18 Concentration effects on SERS conjugates Raman spectra 19
Raman Ocean Optics Data Monday, 2 March 29 Troponin 3 myoglobin 3 25 25 2 2 count 15 count 15 1 1 5 5 7 9 11 13 15 17 19 7 9 11 13 Raman shift(cm -1) 15 17 Count 9 8 7 6 5 4 3 2 1 TROPONIN AND MGLOBIN MIXTURE Laser wavelength 785nm Spectra obtained from Ocean Optics is comparable to mesophotonics system 7 9 11 13 15 17 2
Calibration Test for Ocean Optics Monday, 2 March 29 3 25 myoglobin 1 2 3 4 5 3 25 Isopropanol 1 2 3 2 2 4 count 15 Count 15 5 1 1 5 5 7 9 11 13 15 17 6 8 1 12 14 16 18 21
Summary & Future Activities Monday, 2 March 29 Further investigations with Ocean Optics system e.g concentrations of SERS on resolution, buffers, test line intensity Develop and test a robust multivariate data analysis program for deconvolution of spectra associated with > 4 tags. Find different biomarker and perform multiplexing(6-7plex) assay. Build a portable prototype POC Raman spectrophotometer for lateral flow strip and/or cassettes, to include: - Internal calibration routine. - Test calibration strips. Test the system with other models/application 22
Acknowledgements NPL Robert Porter Smita Thobani James Noble Mateusz Szymanski Simon Attree Maria Lodeiro External Oxonica Ocean Optics Nanosight Thanks to the UK Government for funding the project through the Chemistry and Biology NMS Programme.