1. Detection of exosomal proteins using magnetic particles
Mgr. Jiří Pospíšil Feb. 2018

2. Objectives Exosomes isolation using magnetic particles
Detection of exosomal proteins (survivin)
Biosensor with electrochemical detection for analysis of survivin in body fluids. 2

3. Exosomes Microvesicles particle with size 30 – 100 nm
They are released into extracellular space → presented in all body fluids
György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B,László V, Pállinger E, Pap E, Kittel A, Nagy G, Falus A, Buzás EI. Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles. Cell Mol Life Sci. 2011 Aug;68(16): 3

4. Exosomes
Natasha G, Gundogan B, Tan A, Farhatnia Y, Wu W, Rajadas J, Seifalian AM: Exosomes as Immunotheranostic Nanoparticles. Clin Ther  2014; 36(6):820-9. 4

5. Isolation of exosomes MPs → magnetic particle – 1 mm
-COOH → carboxylate-modified surface
MPs
-COOH

6. Isolation of exosomes MPs → magnetic particle – 1 mm
-COOH → carboxylate-modified surface FC
Anti-CD antibody
MPs
-COOH

7. Isolation of exosomes MPs → magnetic particle – 1 mm
-COOH → carboxylate-modified surface
Antibody – 15 nm
Anti-CD antibodies
MPs
-COOH

8. Isolation of exosomes
Exosomes – 30 – 100 nm

9. Isolation of exosomes MPs → magnetic particle – 1 mm
-COOH → carboxylate-modified surface
Antibody – 15 nm
Exosomes – 30 – 100 nm
MPs
-COOH

10. Isolation of exosomes Modified MPs added to sample (LNCaP, PC3; urine)
Incubation time (exosomes attachment)
MPs with attached exosomes
Washing steps
Lysis of exosomes and releasing magnetic particles
Releasing exosomal content
Removing magnetic particles
Solution with exosomal content (proteins, miRNA, mRNA, DNA)
Picture was made by Ing. Jiří Kudr, Ph.D.

11. Anti-survivin antibodies – functionality
Western blot analysis of LNCaP cell lysate
Monoclonal anti-survivin IgG antibody
Polyclonal anti-survivin IgG antibody
MW (kDa)
100 75 50 37 25 20 15
MW (kDa)
100 75 50 37 25 20 15
10mg 20mg 30mg 40mg
10mg 20mg 30mg 40mg
Data were made by Ing. Vladislav Strimska

12. Electrochemical response of MPs-Ab-AP
Response of magnetic particle conjugated to conjugate antibody-alkaline phosphatase → glassy carbon electrode

13. Electrochemical response of MPs-Ab-AP
Response of magnetic particle conjugated to conjugate antibody-alkaline phosphatase → glassy carbon electrode

14. Thank you for your attention