DNA BıOSENSORS

DNA Biosensors

Biorecognition elements: Nucleic acids

The detection of such diseases via DNA biosensors is generally based on nucleic acid hybridization

Detect: mutations, diseases, pathogens, ions

Robust

Simple

sensitive

Types of recognition elements

Known sequences of nucleic acids (DNA or RNA) are used in nucleic acid biosensors as biorecognition elements

There are some types of probes for development of DNA biosensors; Locked nucleic acids (LNAs), Aptamers Peptide nucleic acids (PNAs) along with natural oligonucleotide orientation.

The hybridization process between the probe fragment and target results in the formation of an output signal

Linear DNA or RNA oligonucleotides • 20-40 bp of single stranded (ss)-DNA or RNA oligonuclotides are generally

used in hybridization processes where complementary base-pairing occur between probe and target.

DNA probes are synthesized by chemical techniques or by PCR

RNA probes are synthesized by RT-PCR to generate cDNAs.

RNA probes can also be generated based on the amino acid sequence of the related protein.

• ssDNA/RNA fragments that is highly selective to its target is immobilized on the electrode surface by protecting the fragment’s reactivity, selectivity, stability and hybridization.

• Hybridization occurs between DNA probe and its complementary strand with the contributions of the weak bonds forming within bases

Hairpin DNA Partially ds-DNA molecule

Has a stem-loop structure due to the hybridization of the certain bases in nucleotide strand

During hybridization with the target molecule, it undergoes a conformational change, and this property is of importance to improve specificity and sensitivity

The free end of the hairpin DNA is generally attached to a label molecule generating a signal upon hybridization.

With hairpin DNA, various biological molecules, such as miRNAs, can be detected.

Locked nucleic acids (LNA) •LNA nucleotides differ from normal DNA nucleotides by containing a methylene bridge between 2’ (oxygen) and 4’ (carbon) positions of the ribose sugar.

•LNA; results in an increase of melting temperature (thermal stability)

resistant to nuclease digestion due to this modification

have low toxicity and high affinity against target probes

sensitive molecules (increase sensitivity)

Peptide nucleic acids (PNA) •modified type of nucleic acids used in DNA biosensors

•synthesized by using a pseudopeptide, which is N-(2aminoethyl)-glycine, in place of sugar phosphate backbone found naturally in DNA/RNA molecules.

•have a neutral backbone, resulting in an increase of binding affinity due to the elimination of repulsion forces

•even single-base mismatches

can be detected as an advantage

of high affinity

•show good recognition and

hybridization properties in

solution.

Aptamers •ss, 40-50 bases of RNA or DNA sequences formed and screened via SELEX (systematic evolution of ligands by exponential enrichment) process.

•have low immunogenicity, high specifity and strong affinity.

•can selectively bind to wide variety molecules; -proteins, - ions,

-toxics, - drugs

-viruses - amino acids,

-inorganic molecules

•One limitation of aptamers is that they are sensitive relative to DNA molecules and so they should be protected from nucleases and high temperature .

Methods used in Nucleic Acid Probe(NAP) immobilization

•As in all biosensor types, immobilization of the biorecognition element onto the transducer is very important in DNA biosensors

•Commonly employed immobilization methods can be classified as; i) adsorption,

ii) covalent binding,

iii) immobilization via biotin-avidin interaction

Adsorption •A simple method to immobilize NAPs onto the transducer, adsorption does not require any modification of the probes

• Immobilization takes place by electrostatic interaction between negatively charged nucleic acid and electrodes modified to have positive charge

•Polymeric films are used in this case to provide interaction with NAPs, with some examples of chitosan, polyaniline and poly-L-lysine.

•Random orientation of the probes is observed with this method which can reduce hybridization efficiency.

Fig. 2. Immobilization of oligonucleotides via electrostatis interaction

Covalent Binding

Covalent binding prevents desorption of the probes from the working electrode surface while providing high binding strength, good orientation and stability

The end of a NAP is grafted on the surface of the working electrode

Types of the covalent binding; Chemisorption

Covalent attachment

Chemisorption •The strong interaction of the thiol group (S-H) with the gold surface, NAPs modified with thiol group have been developed

•Thiolated DNA probes can be directly immobilized onto a gold electrode via strong covalent binding

DNA probe - SH + Au DNA probe - S - Au + e - +

H

+

Fig. 3. Immobilization of oligonucleotide probes onto a gold surface

via strong Au -S interaction

Chemisorption

•Gold nanoparticles (AuNPs) can be used as an interface to carry out chemisorption of NAPs onto the different type of electrodes

•Many kinds of materials as carbon nanotubes, graphene, polyaniline

Fig. 4. Immobilization of oligonucleotide probes onto electrode with the use of nanocomposite materials and AuNPs

Covalent attachment •While probes are modified with thiol group in chemisorption method, covalent attachment utilizes termination of the probes with amine groups (NH2)

•This approach permits immobilization via the covalent binding between amine groups and various functional groups generated on the electrode surface

•Functional groups are carboxyl, aldehyde, epoxy, and sulfonic groups

Fig. 5. Immobilization of oligonucleotide probes by forming functional groups on the electrode surface.

Avidin-biotin interaction

Avidin is a tetrameric protein having size of 70 kDA, it has four binding sites for biotin molecule which can be used to modify the ends of the NAPs

The interaction between avidin and biotin is highly strong as nearly as covalent bonding

Electrode surface can be modified with avidin by two common methods Utilization of the avidin-biotin interaction for immobilization of oligonucleotide probes

Sandwich model consist of biotin -avidin-biotin in immobilization

Fig. 6. Utilization of the avidin-biotin interaction for immobilization

of oligonucleotide probes

Firstly method is carrying out EDC (1-ethyl-3-(3dimethylaminopropyl) carbodiimide)/NHS (N-hydroxysuccinimide) reaction to generate carboxyl groups on the electrode surface and then conjugating avidin molecules on the surface.

NAPs whose 3’ or 5’ end modified with biotin can then be immobilized onto the electrode by affinity binding between biotin and avidin.

Fig. 7. Sandwich model consist of biotin -avidin-biotin in

immobilization

The other method is to employ sandwich model consist of biotinavidin-biotin, respectively.

Biotin is first immobilized onto the electrode surface to bind the avidin proteins instead of forming free carboxyl groups on the electrode surface via EDC/NHC coupling reaction.

Biotin molecules on the electrode surface bind one of the binding sites of avidin while the other binding sites remain free for binding of biotinylated NAPs.

NAPs can be immobilized onto different types of electrodes with this immobilization method.

Transduction Mechanism

• Electrochemical

• Optical

• Piezoelectric

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Table 1 Commonly used materials for fabrication and/or modification of electrodes.

Electrochemical DNA Biosensors

Detection of DNA immobilization by exploiting electrochemical methods: Mikkelsen and Millan

More rapid response

Highly sensitive and selective

Simple and accurate detection

Easy to use

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Electrochemical DNA Biosensors

Principle:

DNA immobilized-electrode

solution containing different DNA sequences

target DNA+probe DNA=ds-DNA on electrode

surface

a change in signal forms.

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Electrochemical DNA Biosensors

Redox active indicator-based detection

Small sized-molecules

Enable reversible exchange of electrons with the electrode

Metal complexes, organic dyes or anticancer agents

Ru(NH3)63+, Ru(bpy)3

3+/2+, Os(bpy)2Cl2, Fe(CN)63-/4-, ferrocene,

methylene blue (MB), meldola’s blue, hematoxylin, ethidium bromide, doxorubicin and daunomycin

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Electrochemical DNA detection mechanisms

They bind ds- or ss-DNA from different positions, and this leads to the classification of redox active indicators into four groups

1.electrostatically bind to the negatively charged phosphate group of DNA

2.bind major or minor groove of the ds-DNA

3.intercalate between G-C base pairs of hybridized DNA

4.bind specifically to ss- or ds-DNA

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Electrochemical DNA detection mechanisms

Enzyme-label based detection

redox active enzymes such as alkaline phosphatase (AIP), glucose oxidase (GOx), and horseradish peroxidase (HRP)

either target or reporter DNA probe is labeled

sandwich method utilizing three nucleic acid sequences is the most common method used

hybridization of the target DNA is provided with both the reporter probe and previously immobilized DNA probe

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Electrochemical DNA detection mechanisms

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Electrochemical DNA detection mechanisms

Label free (Indirect) Detection NAPs or target molecules do not have to be labelled

Because this method based on the intrinsic electroactive property of the nucleic acids

Nucleotides undergo an oxidation-reduction reaction when applying a potential at the electrode

Among four nucleotides, oxidation currents of guanine and adenine are much higher.

Therefore, use of purines results in detection of the hybridization event more easily by monitoring oxidation signals of adenine and guanine

Principle:

ss-DNA probe-target DNA=hybridization

amounts of free A and G in the environment

redox activities of purines

the oxidation signal

to accelerate and amplify the transfer of electrons between electrode and guanine redox mediators suc as Ru(bpy)3

3+ (ruthenium-bipyridine complex) can be used

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Electrochemical DNA detection mechanisms

Optical DNA biosensors •Based optical properties (as a result of the interaction between the optical field and biorecognition element) • Absorbance

• Emission

• Reflection

•The optical DNA biosensors are based on the conversion of base pair recognition into an optical signal

•The measured optical signal is proportional to the concentration of the analysed material

Optical biosensors are commonly divided into two main groups

1. According to the labelling modes; Label-free:

The interaction between the biorecognition element and the transducer generates signal. This signal is detected directly

Label-based modes:

A kind of label is required to produce a measurable signal for the detection

2. According to the detection mechanisms, Surface plasmon resonance (SPR),

Optical fibres,

Colourimetric detection

Surface-enhanced Raman scattering (SERS).

In an SPR-based DNA biosensor;

•the DNA probe is immobilized on the surface of the sensor.

•DNA probe and target molecule interact

•As a result of this interaction, the refractive index is changed and this change is detected by the detector.

•According to the light intensity, the amount of DNA is defined.

Fig. 12. An example of sensogram

Surface plasmon resonance (SPR)

Colourimetric DNA biosensors

• does not require any special equipments

• alternative method for detection of an analyte

• groove binding dyes and fluorescent intercalating are used

• based on the determination of the concentration of a colour reagent in a solution

• only an analytical device is required for the imaging of the used fluorescence or dye. (fluorometer or fluorescence microscope)

• low cost, simplicity, on-site detection, easy preparation, and colourimetric signals can be observed with naked eyes (because of the not necessity of extra instruments)

• nanoparticles and nanorods can be used to enhance the efficiency of detection by their high simplicity and sensitivity.

• The mostly used nanoparticles are gold nanoparticles and quantum dots

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AuNPs • have higher stability and

lower background noise when compared to the fluorescence tagging

• Used to develop an alternative method to labelling for optical DNA detection

Quantum dots • type of nanoparticle using for

fluorescence tagging of biomolecule probe

• The colours of quantum dots are related to their size and emission of wavelength (higher size higher emitted wavelength)

• The quantum dots in different sizes can be used for distinguishing of labels for different targets

(Kuswandi et al., 2005; Teles et al., 2008)

Piezoelectric DNA biosensor Based on measuring changes in the resonance frequency of the resulting piezoelectric crystal with mass change on the crystal surface

There are two main types of piezoelectric devices quartz crystal microbalance (QCM)

A transducer that measure mass

The most electrodes in QCM work are gold electrodes.Because gold is not oxidized in the air, but Cu, Ni, Pt and other metals can also be used.

surface acoustic wave (SAW)

Fig. 14. Experimental apparatus for a piezoelectric sensor (Srivastava et al., 2016).

•QCM is a sensor that is suitable for nucleic acid hybridization detection.

•The specific hybridization between the immobilized DNA/ RNA probe and its complementary sequence in the sample leads to a change in the resonant frequency of the QCM

Fig. 16. Principle of a DNA piezoelectric biosensor (Afzal et al., 2017)

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Applications of DNA Biosensors

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Applications of DNA Biosensors

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Electrochemical biosensor for Mycobacterium tuberculosis DNA detection based on gold nanotubes array electrode platform (Torati et al., 2016)

Electrode Type Gold nanotube array (AuNTsA)

Nucleic Acid Type ss-DNA

Detection Technique CV DPV

Solution

5 mM K3Fe(CN)6/K4Fe(CN)

6 mixtures in 0.05 M PBS solution

20 μM methylene blue solution in PBS

Potential Range 0.6 to -0.4 V. -0.4 to +0.1

Potential Scan Range 10 to 100 mV s-1 -

A Case: Electrochemical DNA Biosensor for Detection of M. tuberculosis

(Torati et al., 2016)

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A Case: Electrochemical DNA Biosensor for Detection of M. tuberculosis

Fig. 17. (A) Demonstration for fabrication of AuNTsA electrode, (B) SEM image of AuNTs.

A

B

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Results: Cyclic Voltammetry

Fig. 18. Cyclic voltagrams of AuNTsA electrode (i), bare Au electrode (ii), AuNTsA/Probe DNA electrode (iii), AuNTsA/Probe/Complementary DNA electrode (iv) in 0.05 M PBS (pH 7.4, 0.15 M NaCl) containing 5 mM [Fe(CN)6]3-/4-.

redox behavior of [Fe(CN)6]3-/4-

(ii): Anodic redox peak in bare Au electrode

(i): large surface area redox peak (iii): limited diffusion of the

redox probe, ssDNA redox peak (iv): increased repellence of

redox species, dsDNA redox peak

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Results: Differential Pulse Voltammetry

Fig. 19. Differential pulse voltagrams of 20 μM methylene blue in 0.05 M PBS (pH 7.4, 0.15 M NaCl) obtained for (i) AuNTsA/Probe DNA, (ii) AuNTsA/Probe /Complementary DNA, (iii) AuNTsA/Probe/Non-complementary DNA electrodes

MB peak current (i): highest peak free guanines (ii): lower peak lower MB accumulation steric hindrance (iii): negligible change free guanines good selectivity

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Results: Differential Pulse Voltammetry

Fig. 20. AuNTsA/Probe DNA electrode after hybridization with various concentrations of complementary DNA

DPV measurements with different concentrations

Linear range: 0.01 ng/ μL -100

ng/μL Detection limit: 0.05 ng/μL

Conclusion • Biosensor are analytical devices that are used for detection of any biogical analyte. In a

DNA biosensor, linear DNA and RNA oligonucleotides, hairpin DNA, locked nucleic acid, peptide nucleic acids, and aptamers are used as biorecognition element.

• There are some immobilization techniques for DNA biosensors, such as adsorption, covalent binding, and avidinbiotin interaction.

• In the electrochemical detection, detection is usually performed via electron transfer between electrode and electroactive indicator or by the intrinsic characteristics of the purine bases.

• By using optical transducer, the desired DNA fragment can be detected based on the optical properties by different detection mechanisms. They are surface plasmon resonance, optical fibers, colourometric detection, and surface-enhanced Raman Scattering.

• The quartz crystal microbalances (QCM) are a transducer that measure mass and the mass change per crystal unit area is directly proportional to the change in frequency

• Applications of the DNA biosensor ranging from pathogen detection to gender detection of some species make it a tremendous technology. With the advancements in the material science and technology, fabrication, modification and implementation of all type of biosensors into daily life is expected to increase.