Detection of exosomal proteins using magnetic particles

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Presentation transcript:

Detection of exosomal proteins using magnetic particles Mgr. Jiří Pospíšil 16 Feb. 2018

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

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):2667-88. 3

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

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

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

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

Isolation of exosomes Exosomes – 30 – 100 nm

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

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.

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

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

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

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