Dual sensor based on gold nanostructured screen-printed carbon electrodes for the detection of prostate specific antigen (PSA)

Transcription

1 Departamento de Química Física y Analítica UNIVERSIDAD DE OVIEDO Dual sensor based on gold nanostructured screen-printed carbon electrodes for the detection of prostate specific antigen (PSA)

2 INTRODUCTION Prostate specific antigen (PSA( PSA) ) is a glycoprotein (93% peptide, 7% sugar) produced by prostate to liquefy the seminal fluid, and is the most reliable tool for diagnosing prostate cancer.. PSA in serum circulates non-complexed (free PSA, fpsa,, MW 33 KDa) and complexed in several forms, being the predominant one the complex with α1- antichymotripsin (PSA-ACT,, MW 9 KDa) Free PSA (fpsa) [tpsa] Total PSA (tpsa) Hidden epitope < 2.5 ng/ml Low cancer risk 2.5 ng/ml - 1 ng/ml Grey zone > 1 ng/ml Cancer risk [fpsa]/[tpsa] >.25 <.25 Low cancer risk Cancer risk Exposed epitopes

3 FUNDAMENTALS Screen-printed Electrodes Nanostructuration Immunological reaction Enzymatic catalysis Voltammetric detection

4 FUNDAMENTALS Screen-printed Electrodes Nanostructuration Screen-printed Carbon Electrode (SPCE) Immunological reaction Enzymatic catalysis 5 μm Screen-printed Gold Electrode (SPGE) Voltammetric detection 1 μm

5 FUNDAMENTALS Screen-printed Electrodes Nanostructuration by synthesis in situ of gold nanoparticles (gold electrodeposition) Nanostructuration S1 7 nm, 1 ng Au Screen-printed Carbon nanogold Electrode (SPC-nAu nau-e) S2 7 nm, 1 ng Au Immunological reaction 1 μm 1 μm S3 7 nm, 7 ng Au S4 16 nm, 7 ng Au S5 16 nm, 185 ng Au Enzymatic catalysis 1 μm 1 μm 1 μm Voltammetric detection

6 FUNDAMENTALS Screen-printed Electrodes Nanostructuration P. Fanjul-Bolado, D. Hernández ndez-santos, M.B. González lez-garcía, a, A. Costa-Garc García, a, Anal. Chem.,, 27 (79( 79), 5272 Metalloenzymatic detection H H H O Immunological reaction - O N - O O P Alkaline Phosphatase N O - Oxidation N O N H Enzymatic catalysis Voltammetric detection O 3-Indoxyl Phosphate i (μa) Ag + Silver ions Metallic silver Silver stripping 3-IP/ IP/Ag IP/ IP/Ag E (V) Indigo Blue

7 IMMUNOSENSOR DEVELOPMENT ANALYTICAL SCHEME a b SPE Pretreatment Adsorption of capture antibody Casein blocking PSA capture Biotinylated detecting antibody Strp- incubation Enzymatic reaction with 3-IP/Ag + Analytical Signal Recording a PSA in sample b Background signal Imunological sensor Analytical detection Immunosensor formats Free PSA Sensor Total PSA Sensor

8 6 DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-PRINTED CARBON Capture Antibody (Free PSA) IMMUNOSENSOR DEVELOPMENT OPTMIZATION OF PAREMETERS AND CALIBRATIONS 6 SPC-nAu nau-e Detecting Antibody 7 Strp i p (μa) ip (μa) ip (μa) [8A6] (μg/ml) [PSA1] (ng/ml) [Strp-] (nm) PARAMETERS SPCEs SPGEs SPC-nAu-Es Capture antibody [8A6] (f), [5G6] (t) (µg/ml) [PSA1] (ng/ml) [Strp-] (nm) CALIBRATIONS SPCE SPGE SPC-nAu-E Linear range (ng/ml) Slope μa/(ng/ml) 5G6 (t) A6 (f) G6 (t) A6 (f) G6 (t) A6 (f) r

9 IMMUNOSENSOR DEVELOPMENT SIMULTANEOUS DETECTION OF fpsa AND tpsa Different coating on each electrode 7 Calibration Plots 6 5 Ag i p (μa) = 4.4 [tpsa] (ng/ml) r =.999 Total PSA Sensor ip (μa) 4 3 Free PSA Sensor total PSA No diffusion of the electroactive product 2 ( ) to the adjacent electrode free PSA 85 1 [PSA] (ng/ml) Total PSA Sensor 75 i p (μa) = 2.53 [fpsa] (ng/ml) r = i (μa) E (V vs. Ag)

10 IMMUNOSENSOR DEVELOPMENT SIMULTANEOUS DETECTION OF fpsa AND tpsa Serum samples Monitor PSA production from three different cultures of human androgen drogen-sensitive LNCaP prostate tumor cells CON LNCaP cells maintained in RPMI 164 culture medium supplemented with 1% Fetal Bovine Serum (FBS) (culture medium with normal content of androgens) FBS (CHT-ST) Charcoal-stripped FBS was used instead of normal complete FBS (culture medium without androgens) DHT Dihidrotestosterone, the active form of testosterone, was used in the culture (culture medium with enhanced content of androgens) 7 Calibration plot of PSA in culture medium tpsa fpsa 6 7 i p (μa) i p (μa) = 4.21 [tpsa] (ng/ml) r =.99 total PSA free PSA i p (μa) = 1.61 [fpsa] (ng/ml) r = [PSA](ng/mL)/number cells x CON FBS(CH-ST) DHT [PSA] (ng/ml) Culture medium

11 Immunobisensor for free and total PSA detection in serum IMMUNOSENSOR DEVELOPMENT SIMULTANEOUS DETECTION OF fpsa AND tpsa ONE-STEP FORMAT a b SPE Pretreatment Adsorption of capture antibody Casein blocking PSA capture Detecting antibody reaction Strp-Biotin reaction Enzymatic reaction with 3-IP/Ag + Analytical Signal Recording a PSA in sample b Background signal free PSA i p (μa) = 4.23 [fpsa] (ng/ml) r =.98 ip (μa) total PSA i p (μa) = 2.91[tPSA] (ng/ml) -.1 r = [PSA] (ng/ml)

12 AUTHORS Vanessa Escamilla GómezG Laura García a Medina Graciela Martínez Paredes Dr. David Hernández ndez Santos Dra. María a Begoña a González García Prof. Agustín n Costa García FINANCIAL SUPPORT Project PC6-4 4

13 Immunobisensor for free and total PSA detection in serum i (μa) E (V vs. Ag) Total PSA Sensor

14 Departamento de Química Física y Analítica UNIVERSIDAD DE OVIEDO Dual sensor based on gold nanostructured screen-printed carbon electrodes for the detection of prostate specific antigen (PSA)