Scholarly article on topic 'Label-free Electrostatic Detection of DNA Amplification by PCR Using Capacitive Field-effect Devices'

Label-free Electrostatic Detection of DNA Amplification by PCR Using Capacitive Field-effect Devices Academic research paper on "Materials engineering"

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Procedia Engineering
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{"field-effect sensor" / "DNA hybridization" / "polymerase chain reaction (PCR)" / "label-free detection" / polyelectrolyte}

Abstract of research paper on Materials engineering, author of scientific article — A. Poghossian, T.S. Bronder, S. Scheja, C. Wu, T. Weinand, et al.

Abstract A capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor modified with a positively charged weak polyelectrolyte of poly(allylamine hydrochloride) (PAH)/single-stranded probe DNA (ssDNA) bilayer has been used for a label-free electrostatic detection of pathogen-specific DNA amplification via polymerase chain reaction (PCR). The sensor is able to distinguish between positive and negative PCR solutions, to detect the existence of target DNA amplicons in PCR samples and thus, can be used as tool for a quick verification of DNA amplification and the successful PCR process.

Academic research paper on topic "Label-free Electrostatic Detection of DNA Amplification by PCR Using Capacitive Field-effect Devices"

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Procedía Engineering 168 (2016) 514 - 517

Procedía Engineering

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30th Eurosensors Conference, EUROSENSORS 2016

Label-free electrostatic detection of DNA amplification by PCR using capacitive field-effect devices

A. Poghossiana,b,*, T.S. Brondera,b, S. Schejaa, C. Wua'e, T. Weinandd, C. Metzger-Boddiend, M. Keusgenc, M.J. Schöninga,b

aInstitute of Nano- and Biotechnologies, FH Aachen, Campus Jülich, D-52428 Jülich, Germany bPeter Grünberg Institute (PGI-8), Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany cInstitute of Pharmaceutical Chemistry, University of Marburg, D-35032Marburg, Germany

dGerbion GmbH & Co. KG, D- 70806 Kornwestheim, Germany eInstitute of Medical Engineering, Xi 'an Jiaotong University School of Medicine, Xi 'an, China

Abstract

A capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor modified with a positively charged weak polyelectrolyte of poly(allylamine hydrochloride) (PAH)/single-stranded probe DNA (ssDNA) bilayer has been used for a labelfree electrostatic detection of pathogen-specific DNA amplification via polymerase chain reaction (PCR). The sensor is able to distinguish between positive and negative PCR solutions, to detect the existence of target DNA amplicons in PCR samples and thus, can be used as tool for a quick verification of DNA amplification and the successful PCR process.

© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.Org/licenses/by-nc-nd/4.0/).

Peer-reviewunder responsibility oftheorganizingcommittee of the30thEurosensors Conference

Keywords: field-effect sensor; DNA hybridization; polymerase chain reaction (PCR); label-free detection; polyelectrolyte

1. Introduction

DNA (deoxyribonucleic acid) biosensors are considered as a very promising tool in many fields of applications ranging from pathogen identification and diagnosis of genetic diseases to drug and food industry [1-3]. Label-free detection of charged molecules (e.g., DNA, proteins) using semiconductor field-effect devices has attracted much attention owing to the well-established technologies available for sensor miniaturization and integration [4-13].

»Corresponding authors: Tel: +49241-6009-53215; fax: +49241-6009-53235. E-mail address: a.poghossian@fz-juelich.de

1877-7058 © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference doi:10.1016/j.proeng.2016.11.512

Recently, we demonstrated the capability of capacitive field-effect EIS (electrolyte-insulator-semiconductor) structures modified with the PAH poly(allylamine hydrochloride) layer for a label-free detection of single- and double-stranded DNA (ssDNA and dsDNA) by their intrinsic molecular charge [14-16]. These sensors can be easily fabricated at low cost and not require complicated chemistry for functionalization of the gate surface and/or probe ssDNA. In the present work, the EIS sensor has been applied for the label-free electrostatic detection of pathogen-specific DNA amplification via standard polymerase chain reaction (PCR).

2. Experimental

2.1. Fabrication of EIS sensor chips

EIS sensor chips (10 mm x 10 mm) consisting of an Al/p-Si/SiO2 structure were fabricated from a p-Si wafer (resistivity: 1-10 Qcm). A 30 nm thick high-quality SiO2 gate oxide was thermally grown by dry oxidation of Si. Then, the rear-side SiO2 layer was etched and subsequently, a 300 nm Al layer was deposited to create an ohmic contact to the p-Si substrate.

2.2. Surface modification steps and measuring setup

In this study, the layer-by-layer technique has been utilized for the adsorption of positively charged PAH macromolecules on the negatively charged SiO2 gate insulator [17] and the immobilization of negatively charged probe ssDNA molecules onto the positively charged PAH layer in accordance with the procedure described in [1416]. The PAH layer was prepared from a 50 ^M PAH solution adjusted with 100 mM NaCl, pH 5.4. For immobilization of 52-mer probe ssDNA, the PAH-modified chip surface was exposed to 5 ^M ssDNA solution for 60 min followed by rinsing step to remove unattached probe ssDNA molecules.

Fig. 1 shows a layer structure of the EIS sensor modified with a PAH layer and measurement setup. The capacitive EIS sensors were characterized in a constant-capacitance (ConCap) mode, which allows direct recording of surface potential changes induced due to each surface modification step (PAH adsorption, probe ssDNA immobilization and complementary target cDNA hybridization). To reduce the influence of the charge-screening effect, the measurements were performed in low-ionic strength solution (0.33 mM phosphate buffer (PBS), 5 mM NaCl, pH 7).

Fig. 1. Layer structure of an EIS sensor modified with a PAH layer and measurement setup. RE: reference electrode (liquid-junction Ag/AgCl

electrode).

3. Results and discussion

The sensor response has been studied after incubation in PCR solutions with/without target DNA of interest (positive/negative PCR). The positive and negative PCR solutions contain extracted template DNA from Mycobacterium paratuberculosis-spiked and non-spiked human sputum, respectively. Prior to on-chip hybridization process, the DNA amplicons were denatured by heating at 95 °C, followed by a rapid cooling step (20 s in ice bath).

Fig. 2 shows the dynamic response of the EIS sensor after different surface modification steps as well as after incubation in positive PCR solution (after 40 amplification cycles). A large potential shift of about 82 mV has been observed after hybridization of PCR-amplified complementary target cDNAs with immobilized probe ssDNA molecules (52-mer). Although an incubation of the PAH/probe ssDNA-modified EIS sensor in negative PCR solution (without target DNA, where no amplification is expected) induces about 42 mV signal change due to the unspecific adsorption of PCR components on the sensor surface (see Fig. 3), a further incubation in positive PCR solution containing amplified target cDNAs results in a sensor signal of 76 mV. Thus, the sensor was able to verify whether PCR is successfully performed or not from the magnitude of signal change.

Fig. 2. Dynamic ConCap response of the EIS sensor after different surface modification steps as well as after incubation in positive PCR solution.

Fig. 3. Dynamic ConCap response of the EIS sensor after different surface modification steps as well as after consecutive incubation in negative

and positive PCR solutions (after 40 amplification cycles).

4. Conclusions

A field-effect capacitive EIS sensor modified with a PAH/ssDNA bilayer has been applied for a label-free electrostatic detection of pathogen-specific DNA amplification by PCR. The sensor can be used for a quick verification of successful PCR processes.

Acknowledgements

This work was supported by the grant from BMBF (project DiaCharge 031A192D).

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