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Departamento de Qumica Fsica y Analtica 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

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SLIDE 1

Dual sensor based on gold nanostructured Dual sensor based on gold nanostructured screen screen-

  • printed carbon electrodes for the

printed carbon electrodes for the detection of prostate specific antigen (PSA) detection of prostate specific antigen (PSA)

Departamento de Química Física y Analítica UNIVERSIDAD DE OVIEDO

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SLIDE 2

INTRODUCTION INTRODUCTION

Free PSA (fPSA) Free PSA (fPSA)

[fPSA]/[tPSA] [fPSA]/[tPSA] < 0.25 < 0.25 Cancer risk Cancer risk > 0.25 > 0.25 Low cancer risk Low cancer risk < 2.5 ng/mL < 2.5 ng/mL Low cancer risk Low cancer risk > 10 ng/mL > 10 ng/mL Cancer risk Cancer risk 2.5 ng/mL 2.5 ng/mL -

  • 10 ng/mL

10 ng/mL Grey zone Grey zone [tPSA] [tPSA]

Total PSA (tPSA) Total PSA (tPSA) Prostate specific antigen ( Prostate specific antigen (PSA PSA) is a glycoprotein ) is a glycoprotein (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 tool for diagnosing prostate cancer diagnosing prostate cancer. PSA in serum . PSA in serum circulates circulates non non-

  • complexed

complexed (free PSA, fPSA, , MW (free PSA, fPSA, , MW 33 KDa) and complexed in several forms, being the 33 KDa) and complexed in several forms, being the predominant

  • ne

the predominant

  • ne

the complex with complex with α α1 1-

  • antichymotripsin

antichymotripsin ( (PSA PSA-

  • ACT

ACT, MW 90 KDa) , MW 90 KDa)

Hidden Hidden epitope epitope Exposed Exposed epitopes epitopes

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 3

FUNDAMENTALS FUNDAMENTALS

Nanostructuration Nanostructuration Enzymatic catalysis Enzymatic catalysis Immunological reaction Immunological reaction Voltammetric detection Voltammetric detection Screen Screen-

  • printed Electrodes

printed Electrodes

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 4

Nanostructuration Nanostructuration Enzymatic catalysis Enzymatic catalysis Immunological reaction Immunological reaction Voltammetric detection Voltammetric detection Screen Screen-

  • printed Electrodes

printed Electrodes

5 5 μ μm m

Screen Screen-

  • printed Carbon Electrode

printed Carbon Electrode (SPCE) (SPCE)

10 10 μ μm m

Screen Screen-

  • printed Gold Electrode

printed Gold Electrode (SPGE) (SPGE)

FUNDAMENTALS FUNDAMENTALS

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 5

Nanostructuration Nanostructuration Enzymatic catalysis Enzymatic catalysis Immunological reaction Immunological reaction Voltammetric detection Voltammetric detection Screen Screen-

  • printed Electrodes

printed Electrodes

Nanostructuration by synthesis Nanostructuration by synthesis in situ in situ of gold nanoparticles

  • f gold nanoparticles

(gold electrodeposition) (gold electrodeposition)

S5 S5 160 nm, 1850 ng Au

1 μm 1 μm

S4 S4 160 nm, 700 ng Au S1 S1 70 nm, 100 ng Au

1 μm

S2 S2 70 nm, 100 ng Au

1 μm

S3 S3 70 nm, 700 ng Au

1 μm

Screen Screen-

  • printed

printed Carbon Carbon – – nanoGold nanoGold Electrode Electrode (SPC (SPC-

  • nAu

nAu-

  • E)

E)

FUNDAMENTALS FUNDAMENTALS

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 6

Nanostructuration Nanostructuration Enzymatic catalysis Enzymatic catalysis Immunological reaction Immunological reaction Voltammetric detection Voltammetric detection Screen Screen-

  • printed Electrodes

printed Electrodes

FUNDAMENTALS FUNDAMENTALS

N H O P

  • O
  • O

O N H O- N H O N H O

Alkaline Phosphatase Ag+

Silver ions

Ag0

Metallic silver

3-Indoxyl Phosphate Indigo Blue Oxidation

N H O P

  • O
  • O

O N H O- N H O N H O

Alkaline Phosphatase Ag+

Silver ions

Ag0

Metallic silver

Ag+

Silver ions

Ag0

Metallic silver

3-Indoxyl Phosphate Indigo Blue Oxidation

  • 20

20 40 60 80 100 120 140 160 0.1 0.2 0.3 0.4 E (V) i (μA)

Silver Silver stripping stripping

3 3-

  • IP/

IP/Ag Ag+

+ + AP

+ AP 3 3-

  • IP/

IP/Ag Ag+

+

  • 20

20 40 60 80 100 120 140 160 0.1 0.2 0.3 0.4 E (V) i (μA)

Silver Silver stripping stripping

3 3-

  • IP/

IP/Ag Ag+

+ + AP

+ AP 3 3-

  • IP/

IP/Ag Ag+

+

  • 20

20 40 60 80 100 120 140 160 0.1 0.2 0.3 0.4 E (V) i (μA)

Silver Silver stripping stripping

3 3-

  • IP/

IP/Ag Ag+

+ + AP

+ AP 3 3-

  • IP/

IP/Ag Ag+

+

Metalloenzymatic detection Metalloenzymatic detection

  • P. Fanjul
  • P. Fanjul-
  • Bolado, D. Hern

Bolado, D. Herná ández ndez-

  • Santos, M.B. Gonz

Santos, M.B. Gonzá ález lez-

  • Garc

Garcí ía, a,

  • A. Costa
  • A. Costa-
  • Garc

Garcí ía, a, Anal. Chem.

  • Anal. Chem., 2007 (

, 2007 (79 79), 5272 ), 5272 DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 7

IMMUNOSENSOR DEVELOPMENT IMMUNOSENSOR DEVELOPMENT

Immunosensor formats Immunosensor formats

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Free PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Free PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

wash wash SPE Pretreatment Adsorption of capture antibody Casein blocking wash wash

Imunological sensor Imunological sensor

3-IP / Ag+ Ag0 AP AP Analytical Signal Recording PSA capture Biotinylated detecting antibody

a b

Strp-AP incubation Enzymatic reaction with 3-IP/Ag+ wash wash wash wash a – PSA in sample b – Background signal 3-IP / Ag+ Ag0 AP 3-IP / Ag+ Ag0 3-IP / Ag+ Ag0 AP AP AP Analytical Signal Recording PSA capture Biotinylated detecting antibody

a b

Strp-AP incubation Enzymatic reaction with 3-IP/Ag+ wash wash wash wash wash wash wash wash a – PSA in sample b – Background signal

Analytical detection Analytical detection

ANALYTICAL SCHEME ANALYTICAL SCHEME

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 8

OPTMIZATION OF PAREMETERS AND CALIBRATIONS OPTMIZATION OF PAREMETERS AND CALIBRATIONS

PARAMETERS PARAMETERS SPCEs SPGEs SPC-nAu-Es Capture antibody [8A6] (f) , [5G6] (t) (µg/mL) 6 10 10 [PSA10] (ng/mL) 300 100 300 [Strp-AP] (nM) 0.5 1 0.5

CALIBRATIONS CALIBRATIONS

Linear range (ng/mL) Slope μA/(ng/mL) r SPCE 5G6 (t) 10 - 100 0.587 0.990 8A6 (f) 10 - 100 0.377 0.999 SPGE 5G6 (t) 10 - 100 0.496 0.990 8A6 (f) 10 - 100 0.277 0.990 SPC-nAu-E 5G6 (t) 1 - 10 12.300 0.994 8A6 (f) 1 - 10 6.081 0.992

10 20 30 40 50 60 150 300 450 600 750 [PSA10] (ng/mL) ip (μA) 10 20 30 40 50 60 70 0.00 0.25 0.50 0.75 1.00 1.25 [Strp-AP] (nM) ip (μA)

10 20 30 40 50 60 5 10 15 20 25 [8A6] (μg/mL) ip (μA)

Capture Antibody Capture Antibody

(Free PSA) (Free PSA)

Strp Strp-

  • AP

AP Detecting Antibody Detecting Antibody

SPC SPC-

  • nAu

nAu-

  • E

E

IMMUNOSENSOR DEVELOPMENT IMMUNOSENSOR DEVELOPMENT

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 9

Free PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Free PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Different coating on Different coating on each electrode each electrode

IMMUNOSENSOR DEVELOPMENT IMMUNOSENSOR DEVELOPMENT

SIMULTANEOUS DETECTION OF fPSA AND tPSA SIMULTANEOUS DETECTION OF fPSA AND tPSA

  • 5

5 15 25 35 45 55 65 75 85 0.1 0.2 0.3 0.4 E (V vs. Ag) i (μA)

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

No diffusion of the electroactive product No diffusion of the electroactive product (Ag (Ag0

0) to the adjacent electrode

) to the adjacent electrode

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) total PSA free PSA

ip (μA) = 4.40 [tPSA] (ng/mL) + 11.32 r = 0.999 ip (μA) = 2.53 [fPSA] (ng/mL) + 0.27 r = 0.991

10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 9 10 11 [PSA] (ng/mL) ip (μA)

Calibration Plots Calibration Plots

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SLIDE 10

SIMULTANEOUS DETECTION OF fPSA AND tPSA SIMULTANEOUS DETECTION OF fPSA AND tPSA Serum samples Serum samples

Monitor PSA production from three different cultures of human an Monitor PSA production from three different cultures of human androgen drogen-

  • sensitive LNCaP prostate tumor cells

sensitive LNCaP prostate tumor cells

CON CON

LNCaP cells maintained in RPMI 1640 culture medium supplemented LNCaP cells maintained in RPMI 1640 culture medium supplemented with 10% Fetal Bovine Serum (FBS) with 10% Fetal Bovine Serum (FBS) (culture medium with normal content of androgens) (culture medium with normal content of androgens)

FBS FBS(CHT

(CHT-

  • ST)

ST)

Charcoal Charcoal-

  • stripped FBS was used instead of normal complete FBS

stripped FBS was used instead of normal complete FBS (culture medium without androgens) (culture medium without androgens)

DHT DHT

Dihidrotestosterone, the active form of testosterone, was used Dihidrotestosterone, the active form of testosterone, was used in the culture in the culture (culture medium with enhanced content of androgens) (culture medium with enhanced content of androgens)

10 20 30 40 50 60 70 [PSA](ng/mL)/number cells x 10 6 CON FBS(CH-ST) DHT Culture medium tPSA fPSA

IMMUNOSENSOR DEVELOPMENT IMMUNOSENSOR DEVELOPMENT

Calibration plot of PSA Calibration plot of PSA in culture medium in culture medium total PSA free PSA

ip (μA) = 4.21 [tPSA] (ng/mL) + 16.40 r = 0.990 ip (μA) = 1.61 [fPSA] (ng/mL) + 2.84 r = 0.990

10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 9 10 11 [PSA] (ng/mL)

ip (μA)

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

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SLIDE 11

wash wash SPE Pretreatment Adsorption of capture antibody Casein blocking wash wash AP wash wash PSA capture Detecting antibody reaction Strp-Biotin reaction wash wash 3-IP / Ag+ Ag0 3-IP / Ag+ Ag0 AP AP Enzymatic reaction with 3-IP/Ag+ Analytical Signal Recording

a b

a – PSA in sample b – Background signal

IMMUNOSENSOR DEVELOPMENT IMMUNOSENSOR DEVELOPMENT

SIMULTANEOUS DETECTION OF fPSA AND tPSA SIMULTANEOUS DETECTION OF fPSA AND tPSA ONE ONE-

  • STEP FORMAT

STEP FORMAT

Immunobisensor for free and total PSA detection in serum Immunobisensor for free and total PSA detection in serum

ip (μA) = 4.23 [fPSA] (ng/mL) + 2.29 r = 0.98 ip (μA) = 2.91[tPSA] (ng/mL) - 0.01 r = 0.998

5 10 15 20 25 30 35 40 45 50 1 2 3 4 5 6 7 8 9 10 11 [PSA] (ng/mL) ip (μA)

total PSA free PSA

slide-12
SLIDE 12

FINANCIAL SUPPORT FINANCIAL SUPPORT Project Project PC06 PC06-

  • 004

004

DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN DUAL SENSOR BASED ON GOLD NANOSTRUCTURED SCREEN-

  • PRINTED CARBON

PRINTED CARBON ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA) ELECTRODES FOR THE DETECTION OF PROSTATE SPECIFIC ANTIGEN (PSA)

Vanessa Escamilla G Vanessa Escamilla Gó ómez mez Laura Garc Laura Garcí ía Medina a Medina Graciela Mart Graciela Martí ínez Paredes nez Paredes

  • Dr. David Hern
  • Dr. David Herná

ández Santos ndez Santos

  • Dra. Mar
  • Dra. Marí

ía Bego a Begoñ ña Gonz a Gonzá ález Garc lez Garcí ía a

  • Prof. Agust
  • Prof. Agustí

ín Costa Garc n Costa Garcí ía a AUTHORS AUTHORS

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SLIDE 13
  • 5

5 15 25 35 45 55 65 75 85 0.1 0.2 0.3 0.4 E (V vs. Ag) i (μA)

Immunobisensor for free and total PSA detection in serum Immunobisensor for free and total PSA detection in serum

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

Total PSA Sensor

3-IP / Ag

+

Ag0 AP 3-IP / Ag

+

Ag0 AP

slide-14
SLIDE 14

Dual sensor based on gold nanostructured Dual sensor based on gold nanostructured screen screen-

  • printed carbon electrodes for the

printed carbon electrodes for the detection of prostate specific antigen (PSA) detection of prostate specific antigen (PSA)

Departamento de Química Física y Analítica UNIVERSIDAD DE OVIEDO