Development Of DNA Biosensor Research Articles

Universal CRISPR/Cas12a-associated aptasensor suitable for rapid detection of small proteins with a plate reader.

With the discovery of the collateral cleavage activity, CRISPR/Cas12a has recently been identified as a key enabling approach in novel DNA biosensor development. Despite its remarkable success in nucleic acid detection, realizing a universal CRISPR/Cas biosensing system for non-nucleic acid targets remains challenging, particularly at extremely high sensitivity ranges for analyte concentrations lower than the pM level. DNA aptamers can be designed to bind to a range of specific target molecules, such as proteins, small molecules, and cells, with high affinity and specificity through configuration changes. Here, by harnessing its diverse analyte-binding ability and also redirecting the specific DNA-cutting activity of Cas12a to selected aptamers, a simple, sensitive, and universal biosensing platform has been established, termed CRISPR/Cas and aptamer-mediated extra-sensitive assay (CAMERA). With simple modifications to the aptamer and guiding RNA of Cas12a RNP, CAMERA demonstrated 100fM sensitivity for targeting small proteins, such as IFN-γ and insulin, with less than 1.5-h detection time. Compared with the gold-standard ELISA, CAMERA achieved higher sensitivity and a shorter detection time while retaining ELISA's simple setup. By replacing the antibody with an aptamer, CAMERA also achieved improved thermal stability, allowing to eliminate the requirement for cold storage. CAMERA shows potential to be used as a replacement for conventional ELISA for a variety of diagnostics but with no significant changes for the experimental setup.

Carbon quantum dots functionalized tapered optical fiber for highly sensitive and specific detection of Leptospira DNA

In this paper, we report on the development of DNA biosensor using carbon quantum dots (CQDs) to enhance Leptospira DNA detection. Leptospira is a bacterium that invades human body and cause leptospirosis disease. Leptospirosis has attracted attention mainly due to several cases of human mortality which have distressed health professionals. This biosensor system employs tapered optical fiber as a sensing transducer. The CQDs were functionalized at the tapered region via in situ deposition (drop-casted) before incubation using commercialized chemicals to link the 16 s ribosomal RNA gene (rrs) probe DNA. Detection was achieved with the occurrence of hybridization between the probe DNA and different concentrations of complementary DNA (Leptospira DNA). This biosensor with limit of detection (LOD) of 1.0 fM demonstrated better sensitivity at 1.8295 nm/nM and higher affinity as its dissociation constant (Kd) of 0.552 × 10−09 M is lower compared to previous reported biosensors without CQDs. High specificity biosensor is validated when minimal shift was observed for non-complementary DNA. This novel biosensor was also able to distinguish between genomic DNA of Leptospira serovars and Clostridium difficile as control sample. The proposed biosensor has successfully exhibited the prominent role of nanomaterial for sensor enhancement. Consequently, the biosensor approach in this study has emphasized the potential for the development of more sensitive and reliable leptospirosis diagnostic testing in the future.

A MoS2 platform and thionine-carbon nanodots for sensitive and selective detection of pathogens

This work focuses on the combination of molybdenum disulfide (MoS2) and à la carte functionalized carbon nanodots (CNDs) for the development of DNA biosensors for selective and sensitive detection of pathogens. MoS2 flakes prepared through liquid-phase exfoliation, serves as platform for thiolated DNA probe immobilization, while thionine functionalized carbon nanodots (Thi-CNDs) are used as electrochemical indicator of the hybridization event. Spectroscopic and electrochemical studies confirmed the interaction of Thi-CNDs with DNA. As an illustration of the pathogen biosensor functioning, DNA sequences from InIA gen of Listeria monocytogenes bacteria and open reading frame sequence (ORF1ab) of SARS-CoV-2 virus were detected and quantified with a detection limit of 67.0 fM and 1.01 pM, respectively. Given the paradigmatic selectivity of the DNA hybridization, this approach allows pathogen detection in the presence of other pathogens, demonstrated by the detection of Listeria monocytogenes in presence of Escherichia coli. We note that this design is in principle amenable to any pathogen for which the DNA has been sequenced, including other viruses and bacteria. As example of the application of the method in real samples it has been used to directly detect Listeria monocytogenes in cultures without any DNA Polymerase Chain Reaction (PCR) amplification process.

Development of an Electrochemical DNA Biosensor Based on Gold Nanoparticles and Thiol Graphene Nanocomposite for Detection of a Specific nuc Gene from Staphylococcus aureus

Background:: An electrochemical DNA biosensor based on gold nanoparticles (AuNPs) and thiol graphene (TGR) nanocomposite modified carbon ionic liquid electrode (CILE) was developed to detect a specific nuc gene of Staphylococcus aureus, which was further used in the analysis of PCR amplification samples from unfrozen pork. Objective:: The development of a DNA biosensor derived from AuNPs-coated TGR could be used as a novel sensing method for the detection of specific ssDNA sequences in biological and clinical samples. Methods: The electrochemical properties of modified CILE electrodes were determined by cyclic voltammetry and electrochemical impedance spectroscopy, and the electrochemical performances of the biosensor were investigated by differential pulse voltammetry. Results:: This gene sensor was able to detect the specific nuc gene from Staphylococcus aureus over the concentration range from 1.0×10-15 mol L-1 to 1.0×10-6 mol L-1 with a limit of detection of 4.5×10-16 mol L-1 (3σ), and it was applied in the detection of Staphylococcus aureus in an unfrozen pork sample after PCR amplification of the nuc gene with satisfactory results. Conclusion:: This gene biosensor showed high sensitivity and good selectivity, wide detection range and low detection limit, which demonstrated an effective tool to detect specific nuc gene sequences of Staphylococcus aureus.

Integration of Nucleic Acid Extraction Protocol with Automated Extractor for Multiplex Viral Detection.

The isolation of nucleic acids (NA) is the preliminary step to carry out genetic studies and DNA biosensor development. The presence of inhibitors in the purified NA interferes with the downstream application. These salts and other organic contaminations particularly challenge the analytical sensitivity of DNA biosensors. The detailed study was carried out to optimize the factors which might affect viral nucleic acid purification. The results suggested that 6 M guanidinium hydrochloride salt concentration was critical for NA isolation. The inverse relation has been found in the pH of the lysis buffer and quality and quantity of NA. The NA yield was relatively stable at pH 4–5. It has been observed that the use of carrier RNA was indispensable for viral genome isolation. The addition of ethanol to lysate in 1:1 ratio greatly improved NA recovery. The elution efficiency of DNase and RNase free water, 1× TE buffer and 1× PCR buffer was compared. The carrier RNA was best eluted in DNase and RNase free water and 1× TE buffer. It was further demonstrated that this method can be automatized for high throughput detection. A simple experiment was conducted to optimize the different parameters of an automated NA extractor to simultaneously extract HBV DNA and HCV RNA. The purified NA was successfully amplified in PCR and RT-PCR to verify the reliability of the established protocol. Thus a semi-automated system for the simultaneous detection of multiple viruses has been demonstrated.

An Electrochemical Study of the Surface Hybridization Process of Morpholino‐DNA: Thermodynamics and Kinetics

AbstractMorpholino (MO) is a neutral analogue of DNA, which shows promise in the development of DNA biosensors and diagnostic devices. The present study explores the hybridization process of a surface‐attached MO 22‐mer with 10‐mer and 20‐mer DNA targets on a gold electrode. The melting process of the MO‐DNA duplex at the electrode/buffer interface is recorded using cyclic voltammetry. These results show that the length of target DNA, the binding location of the target DNA on the surface‐immobilized MO chain, and electrostatic forces from neighbouring duplexes all modulate the stability and hybridization kinetics of the DNA targets with the MO probes. Melting temperatures for immobilized MO‐DNA duplexes are found to be insensitive to ionic strength, provided the duplexes do not have a linker. Although the melting temperature does not shift appreciably with ionic strength, the maximum hybridization yield does. This somewhat surprising observation is considered to originate from an electrostatic limit on the extent of attainable hybridization. It is also reported that hybridization tends to initiate at the upper half of MO probes.

Metallacarboranes as tunable redox potential electrochemical indicators for screening of gene mutation.

The substitution of hydrogen with chlorine in the metallacarborane [3,3-Fe(1,2-closo-C2B9H11)2]- cluster modulates the formal potential of the Fe3+/Fe2+ redox couple, shifting it to a more positive value. Hence, very similar redox probes with a wide range of formal potentials, ranging from negative to positive values, are available. Thus, we have achieved the synthesis and studied the electrochemical behaviour of the sodium salt of [3,3-Fe(8,9,12-Cl3-1,2-closo-C2B9H8)2]- in aqueous media. This strategy allows tuning of the redox potential of the [3,3-Fe(1,2-closo-C2B9H11)2]- framework with a minor change in its shape and dimensions. We also describe the interaction of the prepared [3,3-Fe(8,9,12-Cl3-1,2-closo-C2B9H8)2]- and the pristine [3,3-Fe(1,2-closo-C2B9H11)2]- with DNA. These studies have been carried out not only with DNA in solution but also with DNA immobilized on screen-printed gold electrodes. The results obtained point to a strong interaction between the metallacarboranes and DNA, to a different extent with single stranded DNA (ssDNA) compared to double stranded DNA (dsDNA). This property makes them selective and wide-ranging potential electrochemical indicators of hybridization. The suitability of these new redox indicators for selective DNA biosensor development has been probed by the direct detection of two different mutations associated with cystic fibrosis in PCR amplicons extracted from blood cells.

A comparative study of electrochemical performances of carbon nanomaterial-modified electrodes for DNA detection. Nanotubes or graphene?

A comparative study about the electrochemical response of glassy carbon electrode modified with four different carbon nanomaterial (CNM) against dsDNA is reported. The CNM used were oxidized and nonoxidized multiwalled carbon nanotubes (MWNT-OX and MWNT, respectively), graphene oxide (GO) and chemically reduced graphene oxide (RGO) and dispersed in 3:1 chitosan/water mixture. The electrodes were characterized by cyclic voltammetry, scanning electrochemical microscopy, and contact angle measurements. The results showed that the type and degree of oxidation have a strong effect on the electroactivity of the modified electrode. GCE/RGO clearly exhibited the most electroactive surface among the CNMs, demonstrating that the graphitic structure is highly important. A more sensitive electrochemical response against dsDNA was obtained when GCE/RGO electrode was used. Therefore, RGO is the CNM recommended for further consideration in the development of DNA biosensors.

Simultaneous Electroreduction of Different Diazonium Salts for Direct Electrochemical DNA Biosensor Development

Mixed layers were obtained after one step deposition by reduction of a diazonium salts mixture. The layers are constituted of electroactive 5-hydroxy-1,4-naphthoquinone derivative (JUG), and 4-aminobenzoïc acid (ABA) which allows subsequent grafting of DNA probes. Each functionalizaton step was characterized by IRRAS and XPS. The redox properties of JUG were used to quantify this compound in the film. The combination of surface analysis and voltammetry leads to a quantitative characterization of all the components at each step. The resulting layers are composed of distinct JUG and ABA domains despite the one step deposition procedure. The developed approach allowed rationalization of experimental conditions to design direct electrochemical DNA biosensor with a detection limit of 10pM.

Development of solution-gated graphene transistor model for biosensors

The distinctive properties of graphene, characterized by its high carrier mobility and biocompatibility, have stimulated extreme scientific interest as a promising nanomaterial for future nanoelectronic applications. In particular, graphene-based transistors have been developed rapidly and are considered as an option for DNA sensing applications. Recent findings in the field of DNA biosensors have led to a renewed interest in the identification of genetic risk factors associated with complex human diseases for diagnosis of cancers or hereditary diseases. In this paper, an analytical model of graphene-based solution gated field effect transistors (SGFET) is proposed to constitute an important step towards development of DNA biosensors with high sensitivity and selectivity. Inspired by this fact, a novel strategy for a DNA sensor model with capability of single-nucleotide polymorphism detection is proposed and extensively explained. First of all, graphene-based DNA sensor model is optimized using particle swarm optimization algorithm. Based on the sensing mechanism of DNA sensors, detective parameters (Ids and Vgmin) are suggested to facilitate the decision making process. Finally, the behaviour of graphene-based SGFET is predicted in the presence of single-nucleotide polymorphism with an accuracy of more than 98% which guarantees the reliability of the optimized model for any application of the graphene-based DNA sensor. It is expected to achieve the rapid, quick and economical detection of DNA hybridization which could speed up the realization of the next generation of the homecare sensor system.

Electrochemical DNA biosensor based on the BDD nanograss array electrode

BackgroundThe development of DNA biosensor has attracted considerable attention due to their potential applications, including gene analysis, clinical diagnostics, forensic study and more medical applications. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry in this study.ResultsElectrochemical DNA biosensor was developed based on the BDD film electrode (fBDD) and BDD nanograss array electrode (nBDD). In comparison with fBDD and AuNPs/CA/fBDD electrode, the lower semicircle diameter of electrochemical impedance spectroscopy obtained on nBDD and AuNPs/CA/nBDD electrode indicated that the presence of nanograss array improved the reactive site, reduced the interfacial resistance, and made the electron transfer easier. Using electroactive daunomycin as an indicator, the hybridization detection was measured by differential pulse voltammetry.ConclusionsThe experimental results demonstrated that the prepared AuNPs/CA/nBDD electrode was suitable for DNA hybridization with favorable performance of faster response, higher sensitivity, lower detection limit and satisfactory selectivity, reproducibility and stability.

LbL-assembled multilayer films of dendritic star polymers: surface morphology and DNA hybridization detection

Positively or negatively charged water soluble core–shell star polymers consisting of a dendritic polyphenylene core and an outer flexible shell have been employed for the assembly of multilayer films. The multilayer films were fabricated via the layer-by-layer assembly (LbL) of the oppositely charged dendritic star polyelectrolytes and characterized by surface plasmon resonance spectroscopy (SPR). The assembled multilayer films exhibit morphology changes by undergoing considerable structural reorganization upon post-treatment under different pH conditions. Kinetic binding of probe DNA molecules on the outmost negatively charged surface was studied by SPR as well and showed stable DNA immobilization. DNA hybridization was investigated by surface plasmon field enhanced fluorescence spectroscopy (SPFS), whose hybridization behaviors from one mismatch and complementary target DNA molecules were significantly distinguished. The SPFS based DNA detection on dendritic star polymer multilayers represents a new candidate for the development of DNA biosensors or DNA microarrays.

Mechanistic Studies of Fc‐PNA(⋅DNA) Surface Dynamics Based on the Kinetics of Electron‐Transfer Processes

N-Terminally ferrocenylated and C-terminally gold-surface-grafted peptide nucleic acid (PNA) strands were exploited as unique tools for the electrochemical investigation of the strand dynamics of short PNA(⋅DNA) duplexes. On the basis of the quantitative analysis of the kinetics and the diffusional characteristics of the electron-transfer process, a nanoscopic view of the Fc-PNA(⋅DNA) surface dynamics was obtained. Loosely packed, surface-confined Fc-PNA single strands were found to render the charge-transfer process of the tethered Fc moiety diffusion-limited, whereas surfaces modified with Fc-PNA⋅DNA duplexes exhibited a charge-transfer process with characteristics between the two extremes of diffusion and surface limitation. The interplay between the inherent strand elasticity and effects exerted by the electric field are supposed to dictate the probability of a sufficient approach of the Fc head group to the electrode surface, as reflected in the measured values of the electron-transfer rate constant, k(0). An in-depth understanding of the dynamics of surface-bound PNA and PNA⋅DNA strands is of utmost importance for the development of DNA biosensors using (Fc-)PNA recognition layers.

Conducting polymers with attached platinum nanoparticles towards the development of DNA biosensors

Based on the strong interaction of noble metal particles with the sulphur atoms of thiophene derivatives, platinum nanoparticles (Pt-NPs)-dispersed poly(3-methylthiophene) (P(3-MeTh)) thin films have been successfully prepared. Evidence of the Pt-NPs confinement in the polymer was provided by the electrocatalytic activity of the modified electrodes towards the dissolved oxygen reduction reaction. The efficient immobilization by self-assembly of thiolated oligonucleotides on the embedded Pt-NPs could be monitored by electrochemical quartz crystal microbalance (EQCM) as well as the subsequent hybridization with the complementary sequence. The good conducting properties and high surface area of P(3-MeTh)/Pt-NPs modified electrodes also allowed the detection of both processes by cyclic voltammetry.

MEET THE GUEST EDITOR

Sibel A. Ozkan studied at Ankara University, from where she received her Pharmacy degree in 1985. She obtained her Master’s (1988) and PhD (1994) degrees in the same university, Analytical Chemistry Department. She has become full professor 2003. She has been involved in several analytical chemistry projects related to electroanalytical chemistry and separation techniques on drug active compounds and DNA-Drug interactions. She has organized and participated in organizing committees of a number of international meetings related to Pharmaceutical Sciences (Series of ISOPS-Ankara-Turkey). Her research interest focuses on the electrochemical investigations of pharmaceutically active compounds and their analysis using electroanalytical, chromatographic and spectrophotometric methods, and their validations. Her research area currently moves to the development of DNA biosensors and their interactions with drugs. She is currently a faculty member at the Analytical Chemistry Department at Faculty of Pharmacy, Ankara University.

MEET THE GUEST EDITOR

Sibel A. Ozkan studied at Ankara University, from where she received her Pharmacy degree in 1985. She obtained her Masters (1988) and PhD (1994) degrees in the same university, Analytical Chemistry Department. She has become full professor 2003. She has been involved in several analytical chemistry projects related to electroanalytical chemistry and separation techniques on drug active compounds and DNA-Drug interactions. She has organized and participated in organizing committees of a number of international meetings related to Pharmaceutical Sciences (Series of ISOPS-Ankara-Turkey). Her research interest focuses on the electrochemical investigations of pharmaceutically active compounds and their analysis using electroanalytical, chromatographic and spectrophotometric methods, and their validations. Her research area currently moves to the development of DNA biosensors and their interactions with drugs. She is currently a faculty member at the Analytical Chemistry Department at Faculty of Pharmacy, Ankara University. Prof. Ozkan is active as a referee with Talanta, Electrochimica Acta, Analytica Chimica Acta, Chromatographia, Analytical Letters, Sensor and Actuator B, Biosensors & Bioelectronics, Journal of Separation Science and etc. She is a member of Editorial Board of Chromatographia, Talanta, The Open Analytical Chemistry Journal, The Global Journal of Analytical Chemistry, Journal of Hacettepe University, Faculty of Pharmacy, and Journal of Pharmaceutical Sciences (FABAD). She was Vice Editor of the Special Issue of CHROMATOGRAPHIA Vol. 66, 2007, “Application of Separation Techniques in Turkey” with Dr. Henk LINGEMAN (CHROMATOGRAPHIA Editor). She received Ankara University Scientific Promotion Award in 2003 and Turkish Pharmaceutical Association, Academy of Pharmacy Science Award in 2008. Prof. Ozkan is an author of 124 original and review papers. Some selected publications are cited below.

An Evolution Based Biosensor Receptor DNA Sequence Generation Algorithm

A biosensor is composed of a bioreceptor, an associated recognition molecule, and a signal transducer that can selectively detect target substances for analysis. DNA based biosensors utilize receptor molecules that allow hybridization with the target analyte. However, most DNA biosensor research uses oligonucleotides as the target analytes and does not address the potential problems of real samples. The identification of recognition molecules suitable for real target analyte samples is an important step towards further development of DNA biosensors. This study examines the characteristics of DNA used as bioreceptors and proposes a hybrid evolution-based DNA sequence generating algorithm, based on DNA computing, to identify suitable DNA bioreceptor recognition molecules for stable hybridization with real target substances. The Traveling Salesman Problem (TSP) approach is applied in the proposed algorithm to evaluate the safety and fitness of the generated DNA sequences. This approach improves efficiency and stability for enhanced and variable-length DNA sequence generation and allows extension to generation of variable-length DNA sequences with diverse receptor recognition requirements.

Topographical and Functional Characterization of the ssDNA Probe Layer Generated Through EDC-Mediated Covalent Attachment to Nanocrystalline Diamond Using Fluorescence Microscopy

The covalent attachment method for DNA on nanocrystalline diamond (NCD), involving the introduction of COOH functionalities on the surface by photoattachment of 10-undecenoic acid (10-UDA), followed by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-mediated coupling to NH 2-labeled ssDNA, is evaluated in terms of stability, density, and functionality of the resulting biological interface. This is of crucial importance in DNA biosensor development. The covalent nature of DNA attachment will infer the necessary stability and favorable orientation to the ssDNA probe molecules. Using confocal fluorescence microscopy, the influence of buffer type for the removal of excess 10-UDA and ssDNA, the probe ssDNA length, the probe ssDNA concentration, and the presence of the COOH-linker on the density and functionality of the ssDNA probe layer were investigated. It was determined that the most homogeneously dense and functional DNA layer was obtained when 300 pmol of short ssDNA was applied to COOH-modified NCD samples, while H-terminated NCD was resistant for DNA attachment. Exploiting this surface functionality dependence of the DNA attachment efficiency, a shadow mask was applied during the photochemical introduction of the COOH-functionalities, leaving certain regions on the NCD H-terminated. The subsequent DNA attachment resulted in a fluorescence pattern corresponding to the negative of the shadow mask. Finally, NCD surfaces covered with mixtures of the 10-UDA linker molecule and a similar molecule lacking the COOH functionality, functioning as a lateral spacer, were examined for their suitability in preventing nonspecific adsorption to the surface and in decreasing steric hindrance. However, purely COOH-modified NCD samples, patterned with H-terminated regions and treated with a controlled amount of probe DNA, proved the most efficient in fulfilling these tasks.

Gold nanoparticle-based electrochemical biosensors

The unique properties of gold nanoparticles to provide a suitable microenvironment for biomolecules immobilization retaining their biological activity, and to facilitate electron transfer between the immobilized proteins and electrode surfaces, have led to an intensive use of this nanomaterial for the construction of electrochemical biosensors with enhanced analytical performance with respect to other biosensor designs. Recent advances in this field are reviewed in this article. The advantageous operational characteristics of the biosensing devices designed making use of gold nanoparticles are highlighted with respect to non-nanostructured biosensors and some illustrative examples are commented. Electrochemical enzyme biosensors including those using hybrid materials with carbon nanotubes and polymers, sol–gel matrices, and layer-by-layer architectures are considered. Moreover, electrochemical immunosensors in which gold nanoparticles play a crucial role in the electrode transduction enhancement of the affinity reaction as well as in the efficiency of immunoreagents immobilization in a stable mode are reviewed. Similarly, recent advances in the development of DNA biosensors using gold nanoparticles to improve DNA immobilization on electrode surfaces and as suitable labels to improve detection of hybridization events are considered. Finally, other biosensors designed with gold nanoparticles oriented to electrically contact redox enzymes to electrodes by a reconstitution process and to the study of direct electron transfer between redox proteins and electrode surfaces have also been treated.

Control of DNA self-assembled monolayers surface coverage by electrochemical desorption

In this report we present electrochemical studies of the immobilization of disulfide-modified single-stranded DNA (ssDNA) on gold. The effect of immobilization time on surface coverage was probed by cyclic voltammetry and electrochemical impedance spectroscopy (EIS). Additionally, linear sweep voltammetry was used to perform reductive electrochemical desorption of the ssDNA molecules, and the variations in surface coverage, charge transfer resistance ( R ct) and capacitance ( C) with desorption cycles were monitored by EIS. The results show surface coverages greater than 0.9 for immobilization times of 15 hours or more. The coverage and R ct increased, while the C decreased for higher immobilization times. The opposite trend was observed for an increasing number of desorption cycles. These results suggest that a combination of variations in immobilization time and number of desorption cycles represents an alternative to optimize the density of immobilized DNA strands, which is desirable for the subsequent optimization of DNA hybridization and the development of DNA biosensors.