Multiplexing immunoassay with SERS

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1 Multiplexing immunoassay with SERS Neelam Kumarswami 24th Feb _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 Aim of the project Robust, rapid, low cost, quantitative, multiplexed Point of Care (POC) biosensor platform for early diagnosis of human disease.

3 Monday, 2 March 29 Outline SERS particles for multiplexing and SERS technology Antibody-SERS conjugation Bioactivity of conjugates Raman data 3

4 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

5 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 Intensity is directly proportional to concentration of species nm 785nm plasmons υ R hif ( 1) 5

6 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

7 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

8 Raman spectra for selected SERS Monday, 2 March Intensity Intensity N N D D D D N N D D D D Intensity Intensity N N 5 N 44 N

9 Monday, 2 March 29 Overlay of SERS spectra Regions of variations Intensity R am an shift(cm -1) SERS 42 and 44 chosen for model system studies 9

10 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

11 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

12 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) μg/ml antibody loading 12

13 Characterisation of Conjugates Monday, 2 March 29 Nanosight Dynamic light scattering (DLS) ELISA (activity) Lateral flow assays (activity) Raman UV-Vis 13

14 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 (% ) Size (d.nm) 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 Volume (% ) 1 5 Volume (% ) Size (d.nm) 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

15 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 Size (d.nm) In te n s ity (% ) Size Distribution by Intensity 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 (% ) 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

16 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

17 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

18 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 (42+44)conjugates 16 Intensity Laser wavelength 663 nm

19 Raman Mesophotonics Data(2) 2 (42+44)conjugates conjugates(1/1) conjugates (1/6) 15 Intensity Concentration effects on SERS conjugates Raman spectra 19

20 Raman Ocean Optics Data Monday, 2 March 29 Troponin 3 myoglobin count 15 count Raman shift(cm -1) Count TROPONIN AND MGLOBIN MIXTURE Laser wavelength 785nm Spectra obtained from Ocean Optics is comparable to mesophotonics system

21 Calibration Test for Ocean Optics Monday, 2 March myoglobin Isopropanol count 15 Count

22 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

23 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.

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