Departamento de Química Física y Analítica UNIVERSIDAD DE OVIEDO Dual sensor based on gold nanostructured Dual sensor based on gold nanostructured screen- -printed carbon electrodes for the printed carbon electrodes for the screen detection of prostate specific antigen (PSA) detection of prostate specific antigen (PSA)
DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN- -PRINTED CARBON PRINTED CARBON DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) INTRODUCTION INTRODUCTION Prostate specific antigen (PSA PSA) is a glycoprotein ) is a glycoprotein Prostate specific antigen ( (93% peptide, 7% sugar) produced by prostate to (93% peptide, 7% sugar) produced by prostate to liquefy the seminal fluid, and is the most reliable liquefy the seminal fluid, and is the most reliable tool for diagnosing prostate cancer tool for diagnosing prostate cancer. PSA in serum . PSA in serum circulates non non- -complexed complexed (free PSA, fPSA, , MW (free PSA, fPSA, , MW circulates 33 KDa) and complexed in several forms, being the 33 KDa) and complexed in several forms, being the α 1 α predominant predominant one one the the complex complex with with 1- - antichymotripsin ( antichymotripsin (PSA PSA- -ACT ACT, MW 90 KDa) , MW 90 KDa) Free PSA (fPSA) Total PSA (tPSA) Free PSA (fPSA) Total PSA (tPSA) Hidden Hidden epitope epitope [tPSA] [tPSA] < 2.5 ng/mL 2.5 ng/mL - - 10 ng/mL 10 ng/mL > 10 ng/mL < 2.5 ng/mL 2.5 ng/mL > 10 ng/mL Low cancer risk Low cancer risk Grey zone Grey zone Cancer risk Cancer risk Exposed Exposed [fPSA]/[tPSA] [fPSA]/[tPSA] epitopes epitopes > 0.25 > 0.25 < 0.25 < 0.25 Low cancer risk Low cancer risk Cancer risk Cancer risk
DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN- -PRINTED CARBON PRINTED CARBON DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) FUNDAMENTALS FUNDAMENTALS Screen- -printed Electrodes printed Electrodes Screen Nanostructuration Nanostructuration Immunological reaction Immunological reaction Enzymatic catalysis Enzymatic catalysis Voltammetric detection Voltammetric detection
DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN- -PRINTED CARBON PRINTED CARBON DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) FUNDAMENTALS FUNDAMENTALS Screen- -printed Electrodes printed Electrodes Screen Screen- -printed Carbon Electrode printed Carbon Electrode Screen Nanostructuration Nanostructuration (SPCE) (SPCE) Immunological reaction Immunological reaction 5 μ μ m 5 m Screen- -printed Gold Electrode printed Gold Electrode Screen (SPGE) (SPGE) Enzymatic catalysis Enzymatic catalysis Voltammetric detection Voltammetric detection 10 μ μ m m 10
DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN- -PRINTED CARBON PRINTED CARBON DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) FUNDAMENTALS FUNDAMENTALS Screen- -printed Electrodes printed Electrodes Screen Nanostructuration by synthesis in situ of gold nanoparticles Nanostructuration by synthesis in situ of gold nanoparticles (gold electrodeposition) (gold electrodeposition) S1 S1 S2 S2 70 nm, 100 ng Au 70 nm, 100 ng Au Screen- Screen -printed printed Nanostructuration Nanostructuration Carbon – – nanoGold nanoGold Carbon Electrode Electrode (SPC- (SPC -nAu nAu- -E) E) Immunological reaction Immunological reaction 1 μ m 1 μ m S5 S4 S4 S5 S3 S3 160 nm, 1850 ng Au 160 nm, 700 ng Au 70 nm, 700 ng Au Enzymatic catalysis Enzymatic catalysis 1 μ m 1 μ m 1 μ m Voltammetric detection Voltammetric detection
DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN- -PRINTED CARBON PRINTED CARBON DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) FUNDAMENTALS FUNDAMENTALS Screen- -printed Electrodes printed Electrodes Screen P. Fanjul- -Bolado, D. Hern Bolado, D. Herná ández ndez- -Santos, M.B. Gonz Santos, M.B. Gonzá ález lez- -Garc Garcí ía, a, P. Fanjul A. Costa- -Garc Garcí ía, a, Anal. Chem. Anal. Chem. , 2007 ( , 2007 ( 79 ), 5272 A. Costa 79 ), 5272 Nanostructuration Nanostructuration Metalloenzymatic detection Metalloenzymatic detection H H H H H H O O N N N N N N Immunological reaction Immunological reaction Oxidation Oxidation Alkaline Alkaline N N Phosphatase Phosphatase - O - O O - O - O O O O H H - O - O P P O O Ag + Ag + Ag + Ag 0 Ag 0 Ag 0 Silver ions Silver ions Silver ions Metallic silver Metallic silver Metallic silver 3-Indoxyl 3-Indoxyl Indigo Blue Indigo Blue Enzymatic catalysis Enzymatic catalysis Phosphate Phosphate Silver Silver stripping Silver stripping Silver Silver stripping Silver stripping stripping stripping 160 160 160 140 140 140 120 120 120 + + AP + + AP + + AP 100 100 100 3- 3- 3- 3 3 3 -IP/ -IP/ -IP/ IP/Ag IP/Ag IP/Ag Ag + Ag + Ag + + AP + AP + AP i ( μ A) i ( μ A) i ( μ A) 80 80 80 60 60 60 40 40 40 Ag + Ag + Ag + + + + 3- 3- 3- 3 3 3 -IP/ -IP/ -IP/ IP/Ag IP/Ag IP/Ag 20 20 20 0 0 0 -20 -20 -20 Voltammetric detection Voltammetric detection 0 0 0 0.1 0.1 0.1 0.2 0.2 0.2 0.3 0.3 0.3 0.4 0.4 0.4 E (V) E (V) E (V)
DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN- -PRINTED CARBON PRINTED CARBON DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) IMMUNOSENSOR DEVELOPMENT IMMUNOSENSOR DEVELOPMENT ANALYTICAL SCHEME ANALYTICAL SCHEME 3-IP / Ag + 3-IP / Ag + 3-IP / Ag + 3-IP / Ag + AP AP AP AP AP AP Ag 0 Ag 0 Ag 0 Ag 0 a a b b wash wash wash wash wash wash wash wash wash wash wash wash wash wash wash wash SPE Adsorption of Casein PSA PSA Biotinylated Biotinylated Strp-AP Strp-AP Enzymatic Enzymatic Analytical Analytical a – PSA in sample a – PSA in sample Pretreatment capture blocking capture capture detecting detecting incubation incubation reaction with reaction with Signal Signal b – Background signal b – Background signal 3-IP/Ag + 3-IP/Ag + antibody antibody antibody Recording Recording Imunological sensor Imunological sensor Analytical detection Analytical detection Immunosensor formats Immunosensor formats + + + + + + + + + + 3-IP / Ag 3-IP / Ag 3-IP / Ag 3-IP / Ag 3-IP / Ag 3-IP / Ag 3-IP / Ag 3-IP / Ag 3-IP / Ag 3-IP / Ag AP AP AP AP AP AP AP AP AP AP Ag 0 Ag 0 Ag 0 Ag 0 Ag 0 Ag 0 Ag 0 Ag 0 Ag 0 Ag 0 Free PSA Sensor Free PSA Sensor Total PSA Sensor Total PSA Sensor
DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN- -PRINTED CARBON PRINTED CARBON DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) IMMUNOSENSOR DEVELOPMENT IMMUNOSENSOR DEVELOPMENT OPTMIZATION OF PAREMETERS AND CALIBRATIONS OPTMIZATION OF PAREMETERS AND CALIBRATIONS SPC- SPC -nAu nAu- -E E Capture Antibody Capture Antibody Detecting Antibody Strp- -AP AP Detecting Antibody Strp (Free PSA) (Free PSA) 60 60 70 60 50 50 50 40 40 i p ( μ A) i p ( μ A) 40 i p ( μ A) 30 30 30 20 20 20 10 10 10 0 0 0 0.00 0.25 0.50 0.75 1.00 1.25 0 150 300 450 600 750 0 5 10 15 20 25 [8A6] ( μ g/mL) [PSA10] (ng/mL) [Strp-AP] (nM) Linear range Slope CALIBRATIONS CALIBRATIONS PARAMETERS PARAMETERS r SPCEs SPGEs SPC-nAu-Es μ A/(ng/mL) (ng/mL) Capture antibody [8A6] (f) , [5G6] (t) 6 10 10 5G6 (t) 10 - 100 0.587 0.990 SPCE (µg/mL) 8A6 (f) 10 - 100 0.377 0.999 [PSA10] 300 100 300 5G6 (t) 10 - 100 0.496 0.990 (ng/mL) SPGE 8A6 (f) 10 - 100 0.277 0.990 [Strp-AP] 0.5 1 0.5 1 - 10 12.300 (nM) 5G6 (t) 0.994 SPC-nAu-E 1 - 10 6.081 8A6 (f) 0.992
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