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Copyright Nano Discovery Inc. 2012 Tel: 407-770-8954 One new technology, discover a new world.

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Presentation on theme: "Copyright Nano Discovery Inc. 2012 Tel: 407-770-8954 One new technology, discover a new world."— Presentation transcript:

1 Copyright Nano Discovery Inc Tel: One new technology, discover a new world NanoDLSay for Protein-Protein Interaction Study, Label-free Protein Complex and Protein Oligomer/Aggregate Detection and Analysis in Real Biological Samples

2 Traditional immunoassay assumes proteins only exist as monomer! I. We will not know if any complex is here or not The complex leads to a fake lower concentration II. A fundamental problem with the current bioassay Individual proteinsProtein complexes Protein aggregates The labeled signaling antibody cannot recognize the complexed target protein Conclusion in this case: target protein is not present in the sample at all!!! III. In real biological systems, a biomolecule exists not only as individual molecules, but also as complexes, and this issue is severely neglected by the current assay techniques

3 What is NanoDLSay: Detect target proteins by monitoring the size change of nanoparticles upon binding with the target protein Gold nanoparticles (AuNP) D = 100 nm Protein monomer (~ 5-20 nm) Protein complex (>> 5-20 nm) Immunoglobulin G (IgG) (~ 7-10 nm) D ~ 120 nmD ~ nm D >> nm

4 Dynamic light scattering (DLS): Measure particle size in nanometer size range Scattered light intensity fluctuation Laser beam Scattering light Correlation function Large particle Small particle Average particle size (nm) Intensity Distr. (%)

5 Protein-AuNP interactions: Au-S, Au-N bonding, electrostatic and van der Waals interaction Au 0 Au + Au 0 Au + Protein References 1. Dobrovolskaia MA, Patri AK, Zheng J, Clogston JD, Ayub N, Aggarwal P, Neun BW. Interaction of colloidal gold nanoparticles with human blood: effects on particle size and analysis of plasma protein binding profiles. Nanomedicine (Nanotechnology Biology and Medicine) 2009, 5, Lacerda SHDP, Park JJ, Meuse C, Pristinski D, Becker ML, Karim A, Douglas JF. Interaction of gold nanoparticles with common human blood proteins. ACS Nano 2010, 4, Calzolai L, Franchini F, Gilliland D, Rossi F, Proteinnanoparticle interaction: identification of the ubiquitin-gold nanoparticle interaction site. Nano Lett. 2010; 10: Protein Corona

6 Why not to measure the size of the biomolecules directly using DLS? The scattering light intensity of biomolecules is too weak Such analysis can be done, but at very high concentration – not reflecting the true state of a protein in biological samples Such analysis can only be done on pure protein samples

7 Why Gold Nanoparticles (AuNPs)? Exceptionally intense light scattering property 10 5 times stronger than a fluorescent dye molecule; 100s-1000s times stronger than polystyrene (PS) latex particles Detection limit of DLS for AuNPs can easily reach fM to aM range As an optical probe, AuNPs easily stands out from sample matrix AuNPs Serum A AuNPs PS particle B C Gold nanorods Dark field optical images of AuNPs mixed with human serum (A) and PS particles (B) and gold nanorod AuNR (C)

8 Average particle size increase (nm) Incubation time (min) = 2D of analyte 0 min 30 min At a saturated binding, the average particle size increase of the assay,, is approximately twice of the diameter (D) of the protein A protein complex is typically larger than a monomer A protein complex causes larger average particle size increase of the assay than a protein monomer When a protein exists as an oligomer aggregate, it may crosslink the nanoparticles into clusters, leading to a substantial particle size increase of the assay Applicable for detecting other biomolecule complexes such as DNA-protein complexes NanoDLSay : Detect target proteins in all forms Information on the target molecule is obtained from real biological samples

9 Applications Real-time kinetic binding study of protein- protein interaction Label-free protein complex detection and analysis in real biological samples Label-free protein oligomer and aggregate detection and analysis in real samples Comparison of NanoDLSay with other existing techniques

10 I. Kinetic study of protein-protein interaction Procedure: 1.Immobilize one target A or B protein to the AuNP 2.Mix the target A or B-modified AuNP with target B or A protein 3.Monitor the AuNP size change 4.Binding affinity may be estimated using Langmuir adsorption model Target A Target B Average particle size (nm) Incubation time (min) 0 30 Non-binding proteins Assay format I: immobilize one target protein on AuNP as a probe Requirements: Immobilization of A or B on AuNP does not affect protein-protein interactions Sample suitability: Pure protein samples Homogeneous solution assay, obtain results in minutes Detect both strong and weak binding Monitoring binding in real-time

11 I. Kinetic study of protein-protein interaction Procedure: 1.Mix relevant protein binding partners together 2.Conduct adsorption assay of individual target with AuNP 3.Conduct adsorption assay of mixed product with AuNP 4.Comparison of assay results from 2 and 3 to obtain complex information Assay format II: allow proteins bind first in solution, then conduct adsorption assay + Comparison to obtain binding information Requirements: At least one protein will readily adsorb to AuNP Sample suitability: Pure protein samples The AuNP-adsorption assay does not affect target protein binding Suitable for studying multi-binding partner (more than 2) complexes A B complex Compared to assay format I: + C

12 I. Kinetic study of protein-protein interaction Procedure: 1.Make a target A or B-conjugated AuNP probe 2.Mix the two probes in solution 3.Monitor the size change of the assay 4.A-B interaction leads to AuNP cluster formation Assay format III: two AuNP probe interaction assay Sample suitability: Pure protein samples Increase assay sensitivity for detecting weak interactions Not limited to the size of the proteins or other target molecules Target A Target B + Limitations: Immobilization of A or B on AuNP does not affect protein-protein interactions Compared to assay format I and II:

13 Jans H, Liu X, Austin L, Maes G, Huo Q. Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding study. Anal. Chem. 2009; 81:

14 Austin L, Liu X, Huo Q. An immunoassay for monoclonal antibody isotyping and quality analysis using gold nanoparticles and dynamic light scattering. American Biotechnology Laboratory 2010; 28: 8, Among six monoclonal antibodies from a vendor X, three have quality problem Surface plasmon resonance works on this application, but expensive Assay kit from vendor Y costs $20 per analysis, NanoDLSay costs 20-30¢ per assay

15 II. Label-free protein complex detection and binding partner analysis from real samples Step 1. Determine if a target protein exists as a complex (The final net increase of the AuNP size tells how big the target protein is) Step 2. Screen and identify the binding partners to the target protein Average particle size increase (nm) Incubation time (min) Step 2: Binding partner screening using antibody Step 1: Catch the target Particle size change upon antibody addition c ~ 2D Binding partners Not binding partners

16 Jaganathan, S.; Yue, P.; Paladino, D.C.; Bogdanovic, J.; Huo, Q.; Turkson, J. A functional nuclear epidermal growth factor receptor, Src and Stat3 heteromeric complex in pancreatic cancer cells. PLoS One, 2011, 6(5):e19605 (Open Access). Experiments: 1.Prepare the AuNP immunoprobe for EGFR 2.Use the EGFR-AuNP probe to catch the target 3.Determine if EGFR is in a complex 4.Control: add anti-EGFR to the sample solution, incubate, and then repeat the binding assay with AuNP immunoprobe. ~ 70 nm EGFR is about nm, 70 nm of increase suggests it is a complex! Step 1: Catch the target AuNP immunoprobe for target protein, EGFR Control study

17 Experiments: 1.Add an antibody for the suspected binding partner into the assay solution 2.If the particle size is increased, then it is a positive response, and vice versa 3.Control: add a negative isotype control antibody to the assay solution If we conduct a sandwich assay, the conclusion will be: EGFR is not there! Screening the binding partners in the complex using specific antibody Step 2: Binding partner screening using antibody Negative control

18 III. Label-free protein oligomer/aggregate detection and analysis Average particle size increase (nm) Incubation time (min) = 2D of analyte 0 min 30 min protein monomer oligomers, aggregates o Protein oligomer/aggregates cause AuNP probe cluster formation o Specific detection of target protein oligomer/aggregates in real samples

19 Bogdanovic J, Colon J, Baker C, Huo Q. A label-free nanoparticle aggregation assay for protein complex/aggregate detection and analysis. Anal. Biochem. 2010; 45:

20 Detection of human IgG dimer and discovery of a new molecular test for prostate cancer diagnosis using IgG-AuNP adsorption assay Huo, Q.; Litherland, S.A.; Sullivan, S.; Hallquist, H.; Decker, D.A.; Rivera-Ramirez, I. Developing a nanoparticle test for prostate cancer scoring. J. Translational Medicine, 2012, 10:44 (open access). Citrate-AuNP D ~ 100 nm IgG dimer IgG Average D ~ 300 nm NanoDLSay reveals human IgG dimerization at > 100 µg/mL Tumor-IgG interaction reflected in the IgG-AuNP adsorption assay

21 Comparison of NanoDLSay with other existing techniques

22 NanoDLSay versus Surface Plasmon Resonance (SPR) o Label-free technique o Optical substrate: gold nanoparticle o Read-out: AuNP size change o Homogeneous solution assay o Low cost of consumables o Reveal the size information of the target analyte, distinguish protein complexes and oligomers/complexes from monomers o Label-free technique o Optical substrate: gold thin film o Read-out: refractive index change o Heterogeneous chip assay o High cost of consumables o Does not reveal the size information of the target analyte, does not tell whether a protein is a monomer, complex or oligomer NanoDLSay SPR

23 Comparison of NanoDLSay with co-immunoprecipitation (Co-IP) followed by immunoblotting for protein complex analysis

24 NanoDLSay versus size exclusion chromatography (SEC) and analytical ultra-centrifugation (AU) for protein complex and oligomer/aggregate detection and analysis SEC and AU: o For pure protein solution study only o SEC underestimates complex or oligomer/aggregate formation (eluent dilution disrupts existing complexes/oligomers) o AU overestimates complex or oligomer/aggregate formation (centrifugation artificially increases protein complexes/oligomers) NanoDLSay : o Detect protein complexes, oligomers/aggregates from real samples o Fast screening test for protein complex/oligomer/aggregates

25 Non-specific interactions: effect on Co-IP and NanoDLSay A problem in Co-IP: o Significant non-specific interactions caused by the separation process o The concentration of the particle probes and proteins is artificially increased during centrifugation, increasing non-specific interactions This problem does not exist in NanoDLSay : o The AuNP probe concentration is relatively low, reducing non-specific interactions o No centrifugation separation is involved

26 NDS1200: A new dynamic light scattering instrument designed for performing NanoDLSay Product & Services Automatic measurement of 12 samples Automatic kinetic study of 12 samples Fast analysis time: 10-20s per sample 40 µL assay solution is used for the measurement Low-cost, disposable min-glass tubes with caps are used as sample containers. No cross-contamination between samples High throughput analysis capability: samples/hour The hardware is maintenance-free No special housing environment is required for the instrument Extremely easy-to-use software

27 Product & Services NanoDLSay software: A software designed for flexible, kinetic and high throughput analysis

28 Notes o Patent application pending on NanoDLSay technology and NDS1200 system: PCT/US09/ and PCT/US11/21002 o Nano Discovery Inc. has the exclusive license in the world to practice and commercialize NanoDLSay technology Please Contact Us to Request a Quote: NDS1200 Dynamic light scattering instrument for conducting NanoDLSay Assay kit including disposable sample cells and other consumables 3251 Progress Drive Suite A1 Orlando, FL Phone: NDS-Kit1000 Order Information Or visit online: www. Product and Order Information

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