DNA hybridization electrochemical sensor using conducting polymer
Introduction
DNA diagnostics has become a focus of the biotechnology era (Pividori et al., 2000). Specially, DNA probe assay for the detection of specific base sequences of DNA have been enormous scope of application in biotechnology and medicine, ranging from agriculture and farming to detection of pathogens in foods to genetic diagnostics on human subjects (Hoch et al., 1996).
Currently, popular DNA sequence tests, microarrays for example, are based on fluorescence signals. The polymerase chain reaction (PCR) multiplies tiny amounts of DNA into readable quantities. Although these techniques are extremely sensitive and quantitative, they require time, sample preparation, and expansive equipment. Moreover the systems are so big that they can not be satisfied for the portable point-of-care-test (POCT) application up to now (Wilson, 1998).
DNA Lab-on-a-chips (LOC) are POCT applicable devices that comprise a biological recognition agent (probe single strand DNA, for instance) that confers selectivity, a transducer that provides sensitivity and converts the recognition event into a measurable electronic signal and a microfluidic channel that provides delivery and preparation of sample (Pividori et al., 2000, Lim and Cho, 2000, Service, 1998).
The sequence recognition events depend more on the biological components of sensors in LOC. Thus one key factor in sensor design and construction is the development of tethering technologies for stabilizing biomolecules to surfaces (Lin et al., 2000, Cho et al., 2001).
The immobilization of probe DNA molecules onto solid surfaces is enormous interest in studies of DNA diagnostics. Thus various protocols have been developed to confine oligonucleotides (ODN) and chromosomal DNA onto solid sensors (Wang and Jiang, 2000).
By virtue of possibility of miniaturization and direct detection of electrochemical signal on a noble metal electrode, electrochemical systems have been paid attention to the candidate of LOC sensors. In all electrochemical systems, DNA is bound onto a solid electrode to detect an electrochemical signal that inherently involves surfaces and interfaces of the electrodes. There are a number of examples related electrochemical sensors using conducting polymer (de Lumley-Woodyear et al., 1996, Campbell et al., 2002, Patolsky et al., 2001, Patolsky et al., 2002, Wang and Jiang, 1998, Korri-Youssoufi et al., 1997a, Korri-Youssoufi et al., 1997b, Bäuerle and Emge, 1998). de Lumley-Woodyear et al. introduced water soluble copolymer of acrylamide and vinylimidazole, modified with hydrazine and osmium complex to measure directly as an electrical current. The current flows as a result of continuous electroreduction of H2O2, electrocatalyzed by the horseradish peroxidase label of an oligonucleotide strand when the complementary strand was covalently bound to a hydrogel (de Lumley-Woodyear et al., 1996). Garnier et al. introduced functionalized polypyrrole derivatives that comprise pyrrole monomer for the electrochemical sensing and linker for the immobilization of oligonucleotide. They observed decrease of electrochemical activities due to the change of the size of side group between before and after hybridization (Livache et al., 1994, Godillot et al., 1996, Garnier et al., 1999, Korri-Youssoufi et al., 1997b). Bäuerle et al. introduced nucleobase functionalized polythiophenyl derivatives. They observed changes of absorption maximum and the decrease of the electrochemical activities as well due to the hydrogen bond formation with complementary nucleobases (Bäuerle and Emge, 1998, Emge and Bäuerle, 1997).
This paper shows the procedure used for the novel synthesis of thiophenyl monomer and electropolymerization, the specificity of the immobilization and the ability of the probe oligonucleotide linked to the polythiophenyl compound to be hybridized. We also demonstrate electrochemical phenomena on chip electrode system.
Section snippets
Materials
3-thiophene acetic acid, N-hydroxyphthalimide (NHP), N,N′-dicyclohexylcarbodiimide (DCC), ferrocenyl carboxylic acid (FeCOOH), acetonitrile (99.8%, anhydrous), chloroform, and DMSO were purchased from Aldrich. DNase, RNase, and protease none detected phosphate buffer (pH 7.4) and sodium chloride were purchased from Sigma. An electrochemical grade tetrabutylammonium hexafluorophosphate (TBAHFP) was purchased from Fluka. Target ODNs and amino-modified immobilization probes were purchased from
Characterization of patterned electrochemical chip and its electrochemical phenomena
To investigate performance and consistency of patterned chip, cyclic voltammetric (CV) measurements were performed with the chips in 0.1 M ferrocenyl carboxylic acid in acetonitrile solution (Fig. 1). Fig. 1(a) and (b) show the cyclic voltammogram of Ag/AgCl and Pt pseudo reference electrode system, respectively. It was found from the result that an electrode potential of the Pt pseudo reference electrode was 0.60 V vs. SHE.
Electropolymerization and characterization of PTAE film
The TAE can be electropolymerized in the electrolyte
Conclusion
In summary, the combination of target ODN and probe ODN with conjugated conducting polymers leads to sensitive sequence recognizable hybridization sensor. The next step, the physical and chemical conditions affecting on the sensor response, the combination of microfluidic devices for the precise control of the sample volume and the built-in hybridization sensor are under way in our laboratory. A further improvement in sensitivity and selectivity can also be expected from fully integrated
Acknowledgements
This work was supported by the Ministry of Commerce, Industry and Energy (MOCIE) of the Republic of Korea under the next generation new technology development project (00008069) through the Biochip Project Team at Samsung Advanced Institute of Technology (SAIT).
References (28)
- et al.
Synthesis and molecular recognition properties of DNA- and RNA-base-functionalized oligo- and polythiophene
Synthetic Metals
(1997) - et al.
Toward intelligent polymers: DNA sensors based on oligonucleotide-functionalized polypyrroles
Synthetic Metals
(1999) - et al.
Direct chemical functionalization of as-grown electroactive polypyrrole film containing leaving groups
Synthetic Metals
(1996) - et al.
New method of polypyrrole functionalization toward molecular recognition
Synthetic Metals
(1997) - et al.
Reactive groups on polymer coated electrodes 10. Electrogenerated conducting polyalkylthiophenes bearing activated ester groups
Polymer
(2000) - et al.
Fixation of single-stranded DNA nucleotide by self assembly technology
Colloids and Surfaces A: Physicochemical and Engineering Aspects
(2000) - et al.
Electrochemical genosensor design: immobilisation of oligonuleotides onto transducer surfaces and detection methods
Biosensors and Bioelectronics
(2000) - et al.
Specific recognition of nucleobase-functionalized polythiophenes
Advanced Materials
(1998) - et al.
Enzyme-amplified amperometric sandwich test for RNA and DNA
Analytical Chemistry
(2002) Conducting Polymers, Fundamentals and Applications: A Practical Approach
(1999)
A surface forces study of DNA hybridization
Langmuir
Direct enzyme-amplified electrical recognition of a 30-base model oligonuleotide
Journal of the American Chemical Society
Cyclic Voltammetry as a Tool for Characterizing Conducting Polymers
Nanofabrications and Biosystem
Cited by (106)
Metal-organic frameworks and their derivatives as signal amplification elements for electrochemical sensing
2020, Coordination Chemistry ReviewsCitation Excerpt :In recent decades, as the requirement for ultrasensitive detection has become urgent, researches on improving the response of electrochemical sensing through modification with various functional materials are increasing. So far, numerous micron materials and nanomaterials with diverse features have been applied to electrochemical sensing, containing metal nanoparticles (MNPs) [9,10], carbon nanomaterials [11–13], quantum dots [14,15], polymers [16], and semiconductor materials [17–19]. Thereinto, metal-organic frameworks (MOFs), as porous coordination polymers [20], possess many attractive merits of diverse structures, large specific surface area, high porosity, superior catalytic activity and adjustable physicochemical properties [1,7], which have aroused keen interest of researchers and realized a fast and remarkable progress in the field of electrochemical sensing.
Novel electrochemical genosensor for Zika virus based on a poly-(3-amino-4-hydroxybenzoic acid)-modified pencil carbon graphite electrode
2019, Sensors and Actuators, B: ChemicalCitation Excerpt :One way to immobilize these biomolecules is to use coatings of functionalized polymeric films. Polymeric films that have been used in biosensors include polypyrrole [21,22], polyaniline [23], polythiophene derivatives [24], 2-aminobenzoic acid [25], and 3-hydroxyphenylacetic acid [26], among others. These are excellent materials for the immobilization of biomolecules, since they can be chemically functionalized [20], hence providing desirable functional groups on the surface of the transducer.
Current trends in the development of conducting polymers-based biosensors
2019, TrAC - Trends in Analytical ChemistryElectrochemical aptamer-based sensors for food and water analysis: A review
2019, Analytica Chimica ActaDetection analysis limit of nonlinear characteristics of DNA sensors with the surface modified by polypyrrole nanowires and gold nanoparticles
2018, Journal of Science: Advanced Materials and DevicesLabel-free electrochemical DNA sensor using "click"-functionalized PEDOT electrodes
2015, Biosensors and BioelectronicsCitation Excerpt :The decrease in current intensity observed for electrodes exposed to the HCV-target was attributed to changes in the polymer environment caused by DNA hybridization. It has been reported that the formation of hydrogen bonds after hybridization creates potential barriers that slow down the diffusion of ions into the polymer (Bäuerle and Emge, 1998; Cha et al., 2003; Korri-Youssoufi and Makrouf, 2002; Navarro et al., 2005). These barriers thus reduce the electroactivity and conductivity of the polymer backbone, which is in good agreement with the electrochemical behavior observed.