Stable and Reproducible Indium Tin Oxide Platforms Susana - - PowerPoint PPT Presentation

Electrochemical Detection of Salmonella via On-surface Isothermal Amplification of its Genetic Material onto Highly Stable and Reproducible Indium Tin Oxide Platforms

Susana Barreda-García 1,*, Rebeca Miranda-Castro 1, Noemí de-los-Santos-Álvarez 1 and María Jesús Lobo Castañón 1

1 Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of

Oviedo, Julián Clavería 8, 33006, Oviedo.

* Corresponding author: susanbarreda@gmail.com

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Graphical Abstract

Electrochemical Detection of Salmonella via On-surface Isothermal Amplification of its Genetic Material onto Highly Stable and Reproducible Indium Tin Oxide Platforms

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[Original citation] - Reproduced by permission of The Royal Society of Chemistry

Abstract: Salmonella represents one of the major causes of foodborne diseases in humans,

in addition to provoke important economic losses in the agri-food sector worldwide. Therefore, the surveillance and control of this human pathogenic bacterium in foodstuffs and biological fluids are necessary in order to prevent and diagnose the disease. Traditional culture-based methods require 5 to 6 days to obtain a definitive result. As a faster alternative, herein we report the integration of a nucleic acid-based sensor and an isothermal DNA amplification technique, helicase-dependent amplification (HDA), onto indium tin oxide (ITO) surfaces for the electrochemical/optical detection of a DNA sequence specific for the typA gene of Salmonella. DNA amplification process occurs at 65 ˚C with the reverse primer covalently bound to the ITO surface, whereas forward fluorescein-tagged primer is incorporated in solution. As a result of the isothermal elongation step, fluorescein-tagged DNA duplexes are attached to the ITO surface. Then, an anti-fluorescein-enzyme conjugate is incorporated for subsequent detection of the enzymatic product. This developed integrated sensing platform allows the detection of Salmonella down to 100 genomes in just over 2 hours [1] without need of high-end benchtop instrumentation. Furthermore, the sensing phase maintains its performance even after 9 months storage.

[1] S. Barreda-García, R. Miranda-Castro, N. de-los-Santos-Álvarez, A.J. Miranda-Ordieres, M.J. Lobo-Castañón, Chem. Comm. 53 (2017) 9721-9724.

Keywords: Salmonella; genetic material; helicase-dependent amplification

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Introduction

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Salmonella spp. pathogens constitutes one of the major causes of foodborne diseases in humans worldwide.

Introduction

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The surveillance and control of this human pathogenic bacterium in foodstuffs and biological fluids are necessary in

• rder to prevent and diagnose the disease.

Food Safety Clinical Diagnosing

Introduction

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Traditional microbiological methods for Salmonella detection in food require 5 to 6 working days to obtain a positive result

Amplification Hybridization Detection

Decentralized genetic testing Isothermal nucleic acid amplification + sensing phase thermally stable

Introduction

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HELICASE DEPENDENT AMPLIFICATION (HDA)

Step 3 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Step 2 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ Step 1

65 °C

HDA + electrochemical detection matches real-time PCR

• Biosens. Bieolectron. 68 (2015) 122-128

Anal.Chem. 87 (2015) 8547-8554

• Anal. Bioanal. Chem. 408 (2016) 8603-8610

Helicase DNA polymerase Forward primer Reverse primer SSB protein

Introduction

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To develop a simple and robust platform for the quantification of DNA sequences specific of Salmonella by integrating on-surface HDA and electrochemical detection at indium-tin oxide (ITO) surfaces

Objective

Optimizing the sensing phase formation and the hybridization assay Evaluating the genosensor response and stability Integrating on-surface HDA and electrochemical detection

ITO modification with oligonucleotides

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1. (3-aminopropyl)triethoxysilane (APTES) 2. Sulfosuccinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate 3. Thiolated DNA capture probe (25 mer)

1% APTES

• vernight

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

ITO modification with oligonucleotides

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1. (3-aminopropyl)triethoxysilane (APTES) 2. Sulfosuccinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate 3. Thiolated DNA capture probe (25 mer)

Γactive = 1.7×1014 molecules/cm2

sites 1% APTES

• vernight

Cyclic voltammetry after linking 6-(ferrocenyl)hexanethiol

Quantification

• f Active Sites

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

ITO modification with oligonucleotides

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1. (3-aminopropyl)triethoxysilane (APTES) 2. Sulfosuccinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate 3. Thiolated DNA capture probe (25 mer)

1% APTES

• vernight

ΓssDNA = 2.5×1012 molecules/cm2 Chronocoulometry after interacting with Ru(NH3)6

3+

Quantification

• f bound ssDNA

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

ITO modification with oligonucleotides

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1. (3-aminopropyl)triethoxysilane (APTES) 2. Sulfosuccinimidyl 4-(N-maleimidemethyl)cyclohexane-1-carboxylate 3. Thiolated DNA capture probe (25 mer)

1.5 %

Medium surface density Adequate DNA spacing for hybridization

1% APTES

• vernight

Γactive = 1.7×1014 molecules/cm2

sites

ΓssDNA = 2.5×1012 molecules/cm2

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

Genosensor for Salmonella

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Hybridization efficiency: 50%

Fluorescence Spectroscopy

Genosensor for Salmonella

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Reproducibility: 10 % (5 nM) LOD : 2.5 nM Linear range: 5 to 250 nM

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,2 0,4 0,6 0,8 i / µA E / V vs Ag/AgCl

250 nM 100 nM 25 nM 12.5 nM 5 nM 0 nM y = 2,7082x + 43,6 R² = 0,9989

100 200 300 400 500 600 700 800 100 200 300 inet / nA [Salmonella DNA] / nM

Sensor Response to the Concentration of Salmonella Voltammograms

Genosensor stability

15 5 10 15 20 25 30 25° C 65° C S/B

Thermal Stability

S: 100 nM

Operational Conditions

2 4 6 8 10 12 14 1 120 180 270 S/B Days

Storage Stability

S: 25 nM

2.5% (BSA + Glucose)

100 200 0,4 0,8

i / nA E / V

25 ºC 65 ºC Blank

Dry/ 4 ˚C

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

On-surface HDA amplification

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Function Name Sequence (5’→3’) Forward primer 6-FAM-FP2

6-FAM-GGT CTG CTG TAC TCC ACC TTC AGC

Reverse Primer (solution) RP

TTG GAG ATC AGT ACG CCG TTC T

Reverse primer (immobilized) HS-T10-RP2

HS-C6-(T)10 TTG GAG ATC AGT ACG CCG TTC TGA CGC T

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

On-surface HDA amplification

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Salmonella genome helicase SSB protein polymerase HS-T10-RP2 RP 86bp 6-FAM-amplicon 6-FAM-FP2

• 2. Elongation of anchored RP using as

target 6-FAM-shortened amplicons

• 1. Asymmetric genome amplification

in solution

RP depletion

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

On-surface HDA amplification optimization

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Asymmetry Ratio for primers in solution

500 1000 1500 2000 1:8 1:15 i / nA Primer asymmetry ratio RP:FP

Blank 104 GU

Minimizing non-specific amplification FP: 75 nM RP: lower amount On-surface amplification for 90 min

On-surface HDA amplification optimization

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Amplification time

1 2 3 4 5 75 90 S/B Amplification time / min

RP:FP / 1:15 FP: 75 nM RP: 5 nM

B: Blank S: 104 GU

Analytical performance

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0,0 0,2 0,4 0,6 0,8 1,0 1,2

1,0E+00 1,0E+01 1,0E+02 1,0E+03 1,0E+04 1,0E+05 1,0E+06

Normalized inet Salmonella/GU 1 10 102 103 104 105 106

0,5 1 0,4 0,8 i / µA E / V

Glass-ITO Cu tape Polycarbonate wells Counter electrode Liquid junction Reference electrode

Electrochemical Detection

ITO 1-Naphthyl phosphate DPV 1-Naphtol

AP

RSD = 20 % LOD = 10 GU

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

LOD real-time PCR = 100 GU

Analytical performance

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Log (Salmonella, GU) 1 1.5 2 2.5 3 3.5 4 25 20 15 10 5

j (μA cm-2)

Log (Salmonella, GU) 1 1.5 2 2.5 3 3.5 4 0.8 0.4 1.2

j (μA cm-2)

j (μA cm-2) = 14.45 Log (Salmonella, GU) - 24 R2 = 0.991 j (μA cm-2) = 0.32 Log (Salmonella, GU) – 0.12 R2 = 0.991

45 times higher slope  Highest efficiency

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

Homogeneous amplification + genosensor On-surface amplification

Analytical performance

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Homogeneous amplification + genosensor On-surface amplification

Log (Salmonella, GU) 1 1.5 2 2.5 3 3.5 4 25 20 15 10 5

j (μA cm-2)

Log (Salmonella, GU) 1 1.5 2 2.5 3 3.5 4 0.8 0.4 1.2

j (μA cm-2)

j (μA cm-2) = 14.45 Log (Salmonella, GU) - 24 R2 = 0.991 j (μA cm-2) = 0.32 Log (Salmonella, GU) – 0.12 R2 = 0.991

Reduction of non-specific amplification  Better detectability than real-time PCR

But…

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

Analytical performance

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Optical Detection

RSD = 30%

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4

10 100 1000 10000 100000 1000000

Normalized Abs net Salmonella/GU 10 102 103 104 105 106 Glass-ITO Polycarbonate wells

ITO TMB + H2O2 TMBox

POD Microplate reader λ = 450 nm

LOD = 100 GU

[Original citation] - Reproduced by permission of The Royal Society of Chemistry

Conclusions

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➢ A platform with wide applicability, useful for the quantification of genomic DNA by thiolated oligonucleotides (reverse primer) covalently immobilized on ITO surfaces ➢ It shows excellent thermal and storage stability ➢ Amplification and detection are performed on the same platform without thermal cycling ➢ Electrochemical detection of HDA amplification on ITO electrodes has better limit of detection than optical detection of the same assay ➢ Better detectability than real-time PCR, offering an excellent option for genetic detection at the point-of-need Chemical Communications 53 (2017) 9721-9724 (DOI: 10.1039/C7CC05128J)

Acknowledgments

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Projects CTQ2015-63567-R (MINECO) and FC-15-GRUPIN14-025 (FICYT, Asturias)

• Dr. María Jesús Lobo Castañón
• Dr. Noemí de los Santos Álvarez
• Dr. Rebeca Miranda Castro
• Dr. Susana Barreda García

Ana Díaz Fernández Ramón Lorenzo Gómez Rossella Svigelj Álvaro Sánchez San José