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Oscillographic Polarography of Highly Polymerized Deoxyribonucleic Acid
Abstract
PROCEEDING from my finding1,2 that nucleotides, nucleosides
and the bases of nucleic acids can be analysed by alternating current
oscillographic polarography3-5, I have also tried to study
polymerized deoxyribonucleic acid by this method.
... In this context, the use of electrochemical tec for genetic measurements has been around since 1960. [2] Initially, the electroc activity of the nucleic acid molecules that make up the gene itself was the focus, a tron transfer was studied directly on the electrode. Later, many studies were car ...
... Genetic measurements are important for the rapid diagnosis of infectious diseases, such as coronaviruses, in the field. In this context, the use of electrochemical techniques for genetic measurements has been around since 1960 [2]. Initially, the electrochemical activity of the nucleic acid molecules that make up the gene itself was the focus, and electron transfer was studied directly on the electrode. ...
In this paper, we introduce portable sensors based on genetic measurements that can be used in the field for the diagnosis of infectious diseases and disease risk based on SNPs (single nucleotide polymorphisms). In particular, the sensors are based on electrochemical measurements that can be performed with printed electrodes and small measuring devices. Indicator molecules that can bind to nucleic acid molecules in various ways are already known, and some of these molecules have electrochemical activity. First, we investigated the change in their electrochemical responses in a solution system. As a result, we searched for nucleic acid-binding molecules whose current value changes in the presence of DNA. In addition, when we measured the change in the current value, associated with the amplification of specific genes, such as PCR (polymerase chain reaction) and LAMP (loop-mediated isothermal amplification), we found that the current value decreased with the number of amplifications, indicating that specific genes can be monitored electrochemically. Based on this principle, we showed that pathogenic microorganisms and viruses, such as Salmonella, O157 E. coli, hepatitis B virus, periodontal disease bacteria, antibiotic-resistant bacteria and influenza virus, were able to be measured. The method was also applied to the diagnosis of SNPs, such as ApoE (apolipoprotein E), which is a risk factor for Alzheimer’s disease. Rapid PCR was available with a microfluidic device, and a simple method was also presented with the isothermal amplification of LAMP.
... For a long time, a 1D consideration of these biopolymers prevailed in electrochemistry, even after the deciphering of amino acid sequences and 3D structures of first proteins as well as of a double helical structure of DNA (Nobel Prizes in Chemistry, 1958and 1962, and in Physiology or Medicine, 1962. After the discovery of electrochemical activity of proteins (around 1930 [1,2]) and nucleic acids (around 1960 [3,4]) on mercury electrodes and the registration of reduction of the heme prosthetic group of cytochrome c on mercury, gold, and platinum electrodes without irreversible denaturation of the molecule [5], from the end of 1970 th solids electrodes have come to dominate in studies on proteins [6][7][8][9][10][11] and nucleic acids [12][13][14] electrochemistry. As a result, a great deal of experimental data has been accumulated regarding electrochemical behavior on solid electrodes of proteins via reduction and oxidation of their redox active centers (heme, copper centers, quinones, flavins, or iron-sulfur clusters) and/or oxidation of amino acid residues [15][16][17][18][19][20][21][22][23], as well as of nucleic acids via the oxidation of nitrogenous bases [19,[23][24][25][26]. ...
... In addition, the electrochemical signal of biopolymer due to the oxidation of its monomeric units strongly decreases with the biopolymer molecular weight [36,41]. Based on these principles, electrochemistry allows one to monitor a formation of complexes by 4 biopolymers [42], biopolymer aggregation [43] and degradation [44], as well as post-translational (PTM) or post-replicative (PRM) [45,46] modifications. Such changes in biopolymer structure take place in living systems and are closely connected to various biochemical processes in health and disease [43][44][45][46][47][48][49][50]. ...
The current knowledge regarding direct electrochemistry of proteins and nucleic acids on solid electrodes is reviewed and discussed from the point of view of their 3D structure. The accent on the spatial organization of these biopolymers allows for deeper understanding of their redox transformations and for making some parallels with redox processes in living systems. The 3D concept of biopolymer electrochemistry is presented as having the evident practical potential, enabling to register such molecular events as complex formation, aggregation, and degradation, as well as post-translational or post-replicative modifications of proteins and nucleic acids, which are closely connected to various biochemical processes in health and disease. This mini review aims at drawing attention of bioelectrochemical community to problems related to the interpretation of oxidation signals from biomolecules which are polymeric and possess particular 3D structures. Also, a present lack of systematic studies in terms of ‘structure-properties’ in the field of direct biopolymer electrochemistry, coupled with a deep investigation of reaction mechanisms is emphasized.
... DNA-based detection tools have developed enormously over the years, starting from monitoring the redox reactions of nucleic acid bases, DNAs and their components [3][4][5][6][7][8][9][10][11][12] to extensively reviewed [13][14][15][16][17][18][19][20][21][22][23][24][25] systematic detection of DNA-drug interactions and even lately to monitor Zika and Dengue viruses [26]. Amongst various methods used to detect DNA-drug interactions such as spectrometry, electrophoresis and chromatography, electrochemical techniques play a significant role due to high sensitivity and selectivity, fast response, the possibility to measure low sample concentrations and relatively low equipment costs [13,15,25]. ...
... Although the idea of a DNA sensing dates back to the late 50's in the 20th century and the use of a hanging mercury drop electrode (HMDE) [3,4], the widespread application of genomic sensors based on this idea was only possible when reproducible solid electrodes, often carbon [5][6][7][8][9][10][11] and also metallic (e.g. Au) [12], were introduced, leading to significant progress in DNA research allowing, for instance, the miniaturization of electrodes in microarrays [17] important for simultaneous, fast detection of many samples. ...
For the comparison of the DNA interactions with drugs, two newly synthesized prospective anticancer drugs, 6-(1H-imidazo[4,5-b]phenasine-2-yl)benzene-1,3-diol (IPBD) and, its -Cl derivative (Cl-IPBD) have been compared with doxorubicin, a drug widely used in medicine, and with Vitamin C. These compounds were accumulated at a supercoiled scpUC19 plasmid layer formed on a glassy carbon electrode (GCE). Stability of the drug-plasmid/GCE layer was achieved by initial plasmid accumulation using prolonged potential cycling for ca. 200 min. from highly diluted scpUC19 solutions (8 pg/mL), followed by accumulation of the drugs from 1 µM − 50 µM. Electrochemical properties in terms of the redox potentials of the compounds and capacitative/resistive characteristics of the layers have been tested using, in sequence, four voltammetric methods: Square Wave (SWV), Differential Pulse (DPV) and Alternating Current (ACV) with phase detection 0° and 90°. Importantly, with progressive drug accumulation in the plasmid, for Cl-IPBD, but not for IPBD, an increase in peak (I) at –0.42 V vs. SCE was observed, while biological tests revealed a higher cytotoxic activity for Cl-IPBD vs. IPBD. Moreover, an additional redox signal of Cl-IPBD was observed with the compound reductive accumulation at the plasmid layer in the presence of Vitamin C.
... Therefore electrochemical methods are found to be attractive because these techniques are simple, fast, and low cost and have high sensitivity and selectivity. Electrochemical methods based on the oxidation of GU and UA at unmodified electrodes [3][4][5][6] suffer from disadvantages like high overpotential, sluggish direct electron transfer, poor reproducibility and significant fouling of electrode surface due to adsorption of oxidation products. Therefore the developments of modified electrodes materials to overcome these problems have great importance. ...
... The interferences of different substrates during the simultaneous determination of UA and GU (20 μM UA+ 50 μM GU at pH 6) have been studied under an optimum condition at TiO 2 -600/GP. The commonly interfering agents such as Na + , K + , Ca 2+ , NH 4 + , Fe 2+ , Cl − , NO 3− , CO 3 2− dopamine, epinephrine, adrenaline, ascorbic acid, histamine, serotonin, tyramine, tryptamine, phenylalanine are added in 150-fold and dopamine, epinephrine, adrenaline, ascorbic acid are added 100-fold to the above mixture solution during interference studies. The results show (see ESM, Fig. S5) that there is no significant change of current for the determination is observed (change in current is less than ±9%). ...
Titanium dioxide nanoparticles (NPs) were synthesized by a sol-gel method from hexafluorotitanic acid using poly(ethylene glycol) as a capping agent. The crystal structure and morphology of the NPs were characterized by X-ray diffraction, FESEM, and TEM. The NPs were used to modify a graphite paste electrode for simultaneous determination of uric acid (UA) and guanine (GU). The effect of calcination temperature on crystal structure and electrocatalytic activity was investigated. The electrochemical responses to UA and GU at bare GP, TiO2–350/GP, and TiO2–600/GP electrodes were compared. The DPV oxidation peaks of UA and GU were found to be strongest at around 304 and 673 mV, respectively, against Ag/AgCl reference electrode, and this are well separated for effective simultaneous determination. UA and GU can be simultaneously determined by this method. Response is linear within the range 0.1–500 μM and 0.1–40 μM for UA and GU, respectively. The detection limits are 70 nM for UA and 50 nM for GU (at an S/N ratio of 3). The TiO2–600/GP electrode showed excellent analytical performance when analyzing spiked urine and serum samples.
Graphical abstractA graphical representation of cubic TiO2 nanoparticle formation during hydrolysis through sol-gel process.
... Electrode modifications with electroactive conjugated polymers, such as polypyrrole, polyaniline, or PEDOT functionalized with DNA probes create commercially attractive platforms for NA analysis. For successful polymerization and modification, functionalized polymers can be produced by copolymerization of unmodified and partially modified monomers, such as copolymer poly[(3-acetic acid pyrrole)/(3-N-hydroxyphthalimide pyrrole)], to which amine-terminated DNA DNA biosensor tree depicting the basic principles underlying electrochemical NA sensor designs since (a) the electrochemical activity of NAs at mercury electrodes was demonstrated by Emil Paleček through the use of oscillographic polarography (77). (b) Detection of NAs by direct oxidation (alternatively, by reduction) of NA bases (57,58,71,73). ...
... Below, I consider these and more recently developed label-free concepts(Figure 3g,h). They are all summarized in the biosensor tree that originated from Emil Paleček's pioneering experiments on the electrochemical activity of NAs at mercury electrodes performed back in the late 1950s and early 1960s(77). ...
Sensitive, specific, and fast analysis of nucleic acids (NAs) is strongly needed in medicine, environmental science, biodefence, and agriculture for the study of bacterial contamination of food and beverages and genetically modified organisms. Electrochemistry offers accurate, simple, inexpensive, and robust tools for the development of such analytical platforms that can successfully compete with other approaches for NA detection. Here, electrode reactions of DNA, basic principles of electrochemical NA analysis, and their relevance for practical applications are reviewed and critically discussed.
... The demand for optical and electrical biosensors has grown as alternatives to the current methods, since they can be used as real-time diagnostic devices, requiring no professional handling, with a quick response and relatively low cost of construction, are potentially miniaturized and portable. Recent advances in micro and nanotechnologies have allowed the development of highly sensitive and cost-effective electrochemical and optical biosensors [10] for hepatitis B virus detection [11]. ...
... However, these techniques require the use of expensive equipment and reagents as well as skilled personnel, and are performed exclusively in a hospital or laboratorial environment [18]. Genosensors that do not need an amplification step are of greater interest for public health simplifying and reducing the assay time [11], where the electrochemical methods allows the combination of the especifity of biological identification with the selectivity of physicochemical transduction on the hybridization event [19]. ...
... Such a helpful and quick tool may be electrochemical sensors or biosensors [22]. The first report on electroactivity of DNA bases was published in 1960 [23]. Currently, it is known that electrochemical oxidation signals of guanine (G), adenine (A), cytosine (C), and thymine (T) can be observed on voltammograms [24,25]. ...
The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine.
... With the discovery of the electroactivity of DNA [11], many studies have been carried out for the electrochemical determination of nucleic acids. This initiated the electrochemical determination of drug-DNA interactions and made it possible to electrochemically investigate the interactions of many different types of drugs with DNA ( Figure 1) [10,[12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. ...
The interaction of drugs with DNA is important for the discovery of novel drug molecules and for understanding the therapeutic effects of drugs as well as the monitoring of side effects. For this reason, many studies have been carried out to investigate the interactions of drugs with nucleic acids. In recent years, a large number of studies have been performed to electrochemically detect drug–DNA interactions. The fast, sensitive, and accurate results of electrochemical techniques have resulted in a leading role for their implementation in this field. By means of electrochemical techniques, it is possible not only to demonstrate drug–DNA interactions but also to quantitatively analyze drugs.
In this context, electrochemical biosensors for drug–DNA interactions have been examined under different headings including anticancer, antiviral, antibiotic, and central nervous system drugs as well as DNA-targeted drugs.
An overview of the studies related to electrochemical DNA biosensors developed for the detection of drug–DNA interactions that were reported in the last two decades in the literature is presented herein along with their applications and they are discussed together with their future perspectives.
... The reduction of DNA by electrochemical sensors was reported as early as 1960 [55], and subsequently, since DNA purine bases can be oxidized by electrochemistry, electrochemical sensing methods have been developed for the indirect oxidation of DNA by metal complexes acting as electrochemical mediators [56]. With the development of electrochemical biosensors, nucleic acid has gradually become a bridge between electrode materials and analytes, which also benefits from the easy modification of DNA. ...
Simple Summary
The detection of animal viruses remains a formidable scientific challenge, while concurrently presenting a profoundly consequential practical concern of considerable magnitude, necessitating the development of rapid, sensitive, specific, on-site, cost-effective, and user-friendly diagnostic assays. In response to this demand, electrochemical biosensors have garnered significant attention from researchers. In our review, we comprehensively assess the recent research progress pertaining to electrochemical biosensors for animal viral detection, with a particular focus on the application of screen-printed electrodes.
Abstract
Animal viruses are a significant threat to animal health and are easily spread across the globe with the rise of globalization. The limitations in diagnosing and treating animal virus infections have made the transmission of diseases and animal deaths unpredictable. Therefore, early diagnosis of animal virus infections is crucial to prevent the spread of diseases and reduce economic losses. To address the need for rapid diagnosis, electrochemical sensors have emerged as promising tools. Electrochemical methods present numerous benefits, including heightened sensitivity and selectivity, affordability, ease of use, portability, and rapid analysis, making them suitable for real-time virus detection. This paper focuses on the construction of electrochemical biosensors, as well as promising biosensor models, and expounds its advantages in virus detection, which is a promising research direction.
... Reduction of DNA by electrochemical sensors was reported as early as 1960 [55], and subsequently, since DNA purine bases can be oxidized by electrochemistry, Electrochemical sensing methods have been developed for indirect oxidation of DNA by metal complexes acting as electrochemical mediators [56]. With the development of electrochemical biosensors, nucleic acid has gradually become a bridge between electrode materials and analytes, which also benefits from the easy modification of DNA. ...
Animal viruses are a significant threat to animal health and are easily spread across the globe with the rise of globalization. The limitations in diagnosing and treating animal virus infections have made the transmission of diseases and animal deaths unpredictable. Therefore, early diagnosis of animal virus infections is crucial to prevent the spread of diseases and reduce economic losses. To address the need for rapid diagnosis, electrochemical sensors have emerged as a promising tool. Electrochemical methods present numerous benefits, including heightened sensitivity and selectivity, affordability, ease of use, portability, and rapid analysis, making them suitable for real-time virus detection. This paper focuses on the construction of electrochemical biosensor, as well as promising biosensor models, and expounds its advantages in virus detection, which is a promising research direction.
... They consist of a nucleic acid recognition layer immobilized to the electrochemical transducer. The nucleic acid recognition layer detects changes in DNA structure or the specific sequence of DNA that occurs during the interaction of DNA and binding molecules (2)(3)(4)(5). The interaction of drugs with DNA is one of the main targets in drug discovery. ...
Aim: 1,4-dihydropyridine derivative, 1-(3-phenyl propyl)-4-(2-(2-hydroxybenzylidene) hydrazone)-1,4-dihydropyridine (abbreviated as DHP) was synthesized as potential agent for Alzheimer’s disease which is a progressive neurodegenerative brain disorder affecting millions of elderly people. With this study, the electrochemical properties of DHP were investigated and its interaction with DNA was analyzed by differential pulse voltammetry (DPV) and cyclic voltammetry (CV) measurements. In addition, this study aims to determine degradation mechanism of the DHP molecule by Density-functional theory (DFT) in gas and in aqueous phase. Material and Method: Experimental conditions such as immobilization time, the effect of the scan rate, concentration, and the effect of pH were optimized. The method was validated according to validation parameters such as range, precision, linearity, limit of detection (LOD), limit of quantitation (LOQ) and inter/intraday. Results: Linearity study for the calibration curve of DNA and DHP with DPV was calculated in the calibration range 10-100 µg/mL. The LOD and LOQ values were calculated as 3 and 10 µg/mL and intra-day and inter-day repeatability (RSD %) were 1.85 and 3.64 µg/mL, respectively. After the DHP-DNA interaction, the oxidation currents of guanine decreased as a proof of interaction. The activation energy of the most possible path of reaction was calculated, and their thermodynamically most stable state was determined in gas phase. Conclusion: We developed to improve a sensitive, fast and easy detection process for determination of interaction between DHP and DNA.
... Until almost the late 1950s, nucleic acids were considered electrochemically inert and too large to be analyzed by electrochemistry. In 1958 Professor Emil Paleček found that the nuclear bases adenine and cytosine are electrochemically reduced [4,5]. Typical reduction signals of adenine and cytosine [6] are observable as a signal with a maximum in the region of about -1.30 ...
... This type of sensors can function without the addition of a label; one example is the use of the oxidation signal from guanine for target detection [19]. In 1960 Palecek was the first to use the direct oxidation of guanine on a hanging mercury drop electrode to detect target DNA [21]. ...
In this thesis, we report the use of DNA diluents to reduce surface fouling in peptide-containing, thiol-gold self-assembled monolayers. Two antimicrobial peptides previously developed as therapeutics for Methicillin-resistant Staphylococcus aureus (MRSA) USA300 LAC, P1 and P2, were selected as the peptide probes. Both systems were first tested and verified to form stable monolayers on gold electrode surfaces. Their responses in the presence of two complex biological samples, 50 % simulated nasal mucus and undiluted human serum were recorded. A large reduction in the redox signal from the methylene blue label on the peptide probes was observed, indicating severe surface fouling. To minimize surface fouling, two thiolated DNA diluents, tetra-thymine (T4) and tetra-adenine (A4), were separately incorporated into the P1 and P2 systems, and their responses upon exposure to the two matrices were analyzed. All four systems with a DNA diluent, P1/T4, P1/A4, P2/T4, and P2/A4, showed significantly lower signal reduction, verifying the antifouling capabilities of the two DNA diluents. Although this study’s immediate focus was not on the detection of USA300 LAC, the results are invaluable. It has laid the foundation for future advances in the design and fabrication of electrochemical peptide-based MRSA sensors. Advisor: Rebecca Y. Lai
... Several spectral chromatographic (Li et al., 2003;Zhao et al., 2014), polymerase chain reaction (PCR) assay (Petralia and Conoci, 2017), electrochemical methods (Paleček, 1960;Sassolas et al., 2008;Kato et al., 2008) have come into existence for the detection of DNA bases. Out of these methods, spectral chromatographic and polymerase chain reaction (PCR) assays have shown excellent sensitivity in detecting the DNA. ...
Metal-organic frameworks (MOFs) are broadly known as porous coordination polymers (CP), synthesized by metal-based nodes and organic linkers. MOFs are used in various fields like catalysis, energy storage, sensors, drug delivery etc., due to their versatile properties (tailorable pore size, high surface area, and exposed active sites). This review presents a detailed discussion of MOFs as an electrochemical sensor and their enhancement in the selectivity and sensitivity of the sensor. These sensors are used for the detection of heavy metal ions like Cd²⁺, Pb²⁺, Hg²⁺, and Cu²⁺ from groundwater. Various types of organic pollutants are also detected from the water bodies using MOFs. Furthermore, electrochemical sensing of antibiotics, phenolic compounds, and pesticides has been explored. In addition to this, there is also a detailed discussion of metal nano-particles and metal-oxide based composites which can sense various compounds like glucose, amino acids, uric acid etc. The review will be helpful for young researchers, and an inspiration to future research as challenges and future opportunities of MOF-based electrochemical sensors are also reported.
... DNA detection can be based on the natural electroactivity of the nucleotide residues present in DNA. This idea has been exploited in particular by the Palecek group who showed that DNA is an electroactive compounds and guanine was described as the most redox-active nitrogenous base in DNA (Palecek 1960). The detection of DNA hybridization has attracted considerable attention of chemists and biologists due to its potential applications in gene analysis and clinical diagnosis and pathology (Maire and Ligon 2014;Song et al. 2015;Zappacosta et al. 2015). ...
The development of a simple and inexpensive DNA biosensor based on a gold nanoparticle (Au-NPs)/glutathione (GSH) matrix is reported to characterize DNA hybridization. The adhesion and morphological properties of the film were characterized using contact angle measurements and scanning electronic microscopy (SEM), respectively. The modified gold electrode was characterized by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The results indicate that DNA/AuNPs-GSH/cysteine provides excellent sensitivity for the target DNA in the linear range of response from 7 pM to 5 nM. The interaction of 6-thioguanine (6-tg) with the double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA) modified electrode was investigated using DPV and EIS. Consequently, 6-tg is accumulated onto the modified electrode causing a decrease of the charge transfer resistance (Rct) across the dynamic range from 1 pM to 10 µM. The developed electrochemical DNA biosensor was demonstrated to be efficient to characterize the interactions with drugs and micropollutants in wastewater.
... Most DNA-based electrochemical biosensors for plant pathogen diagnostics involve either label-free or labeldependent voltammetric detection of DNA hybridization (Khater et al., 2017). The earliest published direct electrochemistry using a DNA method was based on oxidation on a Mercury electrode (Paleček, 1960). The coupling of DNAbased analyte capture systems with electrochemical techniques to measure the quantity of capture has been included in the development of fungal plant pathogen diagnostics tools (Drummond et al., 2003;Privett et al., 2010). ...
Plant pathogens are a major reason of reduced crop productivity and may lead to a shortage of food for both human and animal consumption. Although chemical control remains the main method to reduce foliar fungal disease incidence, frequent use can lead to loss of susceptibility in the fungal population. Furthermore, over-spraying can cause environmental contamination and poses a heavy financial burden on growers. To prevent or control disease epidemics, it is important for growers to be able to detect causal pathogen accurately, sensitively, and rapidly, so that the best practice disease management strategies can be chosen and enacted. To reach this goal, many culture-dependent, biochemical, and molecular methods have been developed for plant pathogen detection. However, these methods lack accuracy, specificity, reliability, and rapidity, and they are generally not suitable for in-situ analysis. Accordingly, there is strong interest in developing biosensing systems for early and accurate pathogen detection. There is also great scope to translate innovative nanoparticle-based biosensor approaches developed initially for human disease diagnostics for early detection of plant disease-causing pathogens. In this review, we compare conventional methods used in plant disease diagnostics with new sensing technologies in particular with deeper focus on electrochemical and optical biosensors that may be applied for plant pathogen detection and management. In addition, we discuss challenges facing biosensors and new capability the technology provides to informing disease management strategies.
... By the time being, a lot of effort has been gone into direct and catalytic detection of nucleic acid molecules based on the electrochemical properties of nucleotide residues, namely their nitrogenous bases and sugars, on various electrodes (mercury, copper, carbon, gold) [23][24][25][26][27][28][29] . Interestingly, that the electrocatalytic oxidation of sugars within single-and double-stranded DNA (ssDNA and ds-DNA) on copper electrodes leads to the response proportional to the nucleic acid strand thickness and length (to the number of sugar moieties) [28] . ...
The effect of Prussian Blue (PB, ferric hexacyanoferrate) on oxidation of free nucleobases, synthetic oligonucleotides, single- and double-stranded DNA (ssDNA and dsDNA) was evaluated by cyclic voltammetry (CV) and flow injection analysis (FIA) on carbon screen printed electrodes, both bare (SPE) and PB modified (SPE/PB). It has been found that electrocatalytic oxidation of nucleobases, namely guanine, adenine, thymine, and cytosine, or nucleobase residues takes place via electrochemically generated Berlin Green (BG, fully oxidized form of PB). The constant potential FIA allowed one to register direct electrooxidation of all DNA nitrogenous bases at 0.95 V and three nitrogenous bases (except for cytosine) at 0.70 V on SPE and SPE/PB. The modification of electrode surface with PB resulted in a shift of oxidation potential to less positive values and enhanced (up to 8 times) detection sensitivity for DNA nucleobases. The pronounced catalytic effect of electrogenerated BG on their oxidation has also been observed for synthetic oligonucleotides and degraded ssDNA of natural origin. By fractionating ssDNA, it has been found that the main contributor to the total oxidation signal is the fraction of lowest molecular weight (< 3 kDa). At the same time, no oxidation signal was observed for large molecular weight native dsDNA in the potential range from 0 to 1 V on bare SPE and SPE/PB in both CV and FIA measurement modes. Therefore, the advanced electrochemical detection by FIA with SPE/PB was achieved for all nucleobases, synthetic oligonucleotides, and ssDNA of natural origin. Moreover, the obtained results expand the contemporary understanding of DNA electrochemistry.
... The development of simple and sensitive determination of DNA with the specific sequence is of considerable practical interest for many years. Electrochemical behavior of the direct electron transfer of nucleobases was well studied by using the mercury [1], boron-doped diamond [2,3] and diamond-like carbon electrode [4]. ...
... Palecek et al. [181] was first to propose a method to distinguish between double stranded and single stranded DNA through reduction. Later, the same group placed an electrochemical technique to show a distinguishing electrochemical signal between ssDNA and dsDNA by adsorptive stripping voltammetry [185,186]. The electroactive nature of the purine and pyrimidine bases at the electrodic system makes a system to carry out electrocatalytic analysis to study the oxidation or reduction [187]. ...
The graphene is a highly preferable material among all carbon materials and it acquired considerable significance owing to its extraordinary properties. However, this chapter offers a special focus on the preparation methods, structure, properties and applications of graphene, graphene oxide (GO), and graphene‐related materials (GRM). In particular, the GRMs like carbon nanotubes provided lot of benefits to medicinal fields. The importance of this aspect is described in brief. Apart from these, the thermal stability (physical property) is illustrated via the phonon behavior after applying the thermal energy to the GRM system. Furthermore, the electronic properties are discussed at length due to having an exposure of outstanding electronic and transport behavior of charges. In addition, an aspect like how these graphene‐based materials are equipped as advanced transistors, FETS, inverters, and integrated circuits is explained.
... To know more about DNA structures and its molecular charge readers are requested to consult [4], [5], [6]. Nucleotides are electroactive compounds that produce reduction and oxidation signals after hybridization [7]. These high electrostatic potentials can be exploited with VLSI charge sensitive electronic structures. ...
The need for rapid, accurate sequencing of the deoxyribonucleic acid (DNA) molecule has been a major goal of biomedical research and has important implications for human health. The term sequencing is used to describe the elucidation of the basic components of the molecule, which exists as a variable length chain of four basic nucleotides (the component molecules of DNA) including a phosphate group, a sugar, and one of four different nitrogenous bases. The overall molecular structure of DNA is negatively charged and has a negative electrostatic potential due to the negatively charged phosphate backbone. The nucleotides are electroactive compounds that produce reduction and oxidation signals after hybridization. These high electrostatic potentials can be exploited with VLSI charge sensitive electronic structures. Novel methods by which DNA sequencing can be done in more accurate, high-throughput, and faster ways are in development and here, a novel device for sequencing using Gate-All-Around (GAA) nanowire MOSFET device is presented.
... Most of the label-free electrochemical methods for DNA detection rely on the intrinsic electroactivity of DNA purine bases [8]. The first assay of label-free DNA electrochemical detection, developed by Paleček 60 years ago, discriminated ssDNA versus dsDNA through direct reduction of DNA bases at a mercury electrode [86]. Among the four nucleobases, guanine and adenine were found to be the most easily oxidized at carbon electrodes [87]. ...
Nowadays, it has become clear that the critical demands of biosensor development can only be achieved with advanced nanoscale materials. The unique electronic and structural properties of carbon nanomaterials (CNs) and the recent advances in their design and synthesis methodology have enabled new high-performance electrochemical sensing platforms. Furthermore, the progress made towards the functionalization of these nanomaterials led to successful interfacing of biomolecules, such as DNA, and thus to an extensive development of CNs-based electrochemical DNA sensors. The transduction of DNA hybridization events into electrical signals has facilitated the development of rapid, highly sensitive, and highly specific sensing devices employed in important fields such as medical diagnosis, food analysis, and environmental monitoring. A broad range of CNs such as two-dimensional graphene, one-dimensional carbon nanotubes, and zero-dimensional graphene or carbon quantum dots are being explored for designing high-performance DNA-sensing devices. This chapter provides a comprehensive overview of the recent strategies for CNs incorporation into novel DNA-sensing schemes for medical, food, and environmental applications. Likewise, the issues required for an extensive implementation of CNs-based electrochemical DNA sensors in POC technology are critically presented.
... To know more about DNA's structures and its molecular charge, readers are requested to consult [4][5][6]. Nucleotides are electroactive compounds that produce reduction and oxidation signals after hybridization [7]. These high electrostatic potentials can be exploited with VLSI charge-sensitive electronic structures. ...
DNA sequencing is a critical functionality in biomedical research, and technical advances that improve it have important implications for human health. Novel methods by which sequencing can be accomplished in more accurate, high-throughput, and faster ways are in development. Here, we review VLSI biosensors for nucleotide detection and DNA sequencing. Implementation strategies are discussed and split into function-specific architectures that are presented for reported design examples from the literature. Lastly, we briefly introduce a new approach to sequencing using Gate All-Around (GAA) nanowire Metal Oxide Semiconductor Field Effect Transistors (MOSFETs) that has significant implications for the field.
... Since the discovery of the electroactivity of DNA bases [14], various strategies have been investigated for the improvement of the sensitivity and selectivity of electrochemical DNA detection. Unfortunately, there is usually a trade-off between specificity and sensitivity. ...
Applications of molecular techniques to elucidate identity or function using biomarkers still remain highly empirical and biosensors are no exception. In the present study, target-specific oligonucleotide probes for E. coli K12 were designed thermodynamically and applied in an electrochemical DNA biosensor setup. Biosensor was prepared by immobilization of a stem–loop structured probe, modified with a thiol functional group at its 5’ end and a biotin molecule at its 3’ end, on a gold electrode through self-assembly. Mercaptopropionic acid (MPA) was used to optimize the surface probe density of the electrode. Hybridization between the immobilized probe and the target DNA was detected via the electrochemical response of streptavidin-horseradish peroxidase in the presence of the substrate. The amperometric response showed a linear relationship with the target DNA concentration, ranging from 10 and 400 nM, with a correlation coefficient of 0.989. High selectivity and good repeatability of the biosensor showed that the thermodynamic approach to oligonucleotide probe design can be used in development of electrochemical DNA biosensors.
... In the 1960s, the electrochemical activity of highly polymerized deoxyribonucleic acid was found by oscillographic polarography (Palecek, 1960). In recent years, research on properties of nucleic acid on the electrode surface (Ferapontova, 2017), modification of electrode surface (Ji et al., 2019), characteristics of nucleic acid hybridization made some progress. ...
Efficient and rapid detection of pathogens plays an important role in food safety, disease prevention, diagnosis and environmental monitoring. The traditional method for pathogen detection is plate culturing, consuming lots of time on separating, culturing and identifying pathogens by morphological characteristics, biochemical and serological reactions. It is a great advantage to take nucleic acids of pathogens as targets for detection because of higher specificity. The polymerase chain reaction (PCR) greatly shortens the time of pathogen detection but it is heavily dependent on temperature control instruments. Although isothermal amplification overcomes the defects of temperature control, it requires multiple enzymes or complex primers. Here, we summarize the recent advances in the amplification free methods for pathogen detection which are well developed for their simplicity, sensitivity and rapidity. Without nucleic acid amplification, we can directly detect the original nucleic acids of the samples rather than amplified nucleic acids. The amplification free methods for nucleic acid detection are mainly classified into electrochemical biosensors, optical biosensors and piezoelectric plate biosensors. This article describes the principles and compares the advantages and disadvantages of these methods. We further discuss the challenges and directions of this field, providing an overview for future researchers.
... After that, Ko et al. synthesized carboxylic acid functionalized PPy nanotubes and grafted ssDNA to the nanotubes by covalent bond [88]. Both DNA and RNA have been verified to generate redox signals after hybridization based on their electroactive compounds [89]. However, for a long time, it was difficult to detect the signals with a dsDNA biosensor. ...
Electrical phenomenon is ubiquitous in any biological system. However, most synthetic biomaterials are insulators to either electrical or ionic current. To mimic the electrical and ionic conductivities of natural tissues, electrically conductive polymers have been studied and are becoming a new class of biomaterials. This chapter focuses on polypyrrole, one of the most widely investigated synthetic and intrinsically conductive polymers. Polypyrrole is a heterocyclic polymer that is both electrically conductive and ionically active. It can be easily synthesized through electrochemical polymerization or oxidative polymerization. Because of its unique properties, polypyrrole has been studied for sensing, drug delivery, and actuation. Because of its good biocompatibility, it has been used to interface electrical elements and tissues, either for recording or stimulation purpose. Polypyrrole can also be chemically modified to carry functional groups and biomolecules, allowing both specific biological recognition and electrical stimulation. This chapter also discusses a unique soft polypyrrole membrane that can be easily used as biomaterials. Hopefully, the readers of this chapter would appreciate the importance of electrical conductivity for biomaterials and the usefulness of polypyrrole.
Due to their life cycle, viruses can disrupt the metabolism of their hosts, causing diseases. If we want to disrupt their life cycle, it is necessary to identify their presence. For this purpose, it is possible to use several molecular-biological and bioanalytical methods. The reference selection was performed based on electronic databases (2020–2023). This review focused on electrochemical methods with high sensitivity and selectivity (53% voltammetry/amperometry, 33% impedance, and 12% other methods) which showed their great potential for detecting various viruses. Moreover, the aforementioned electrochemical methods have considerable potential to be applicable for care-point use as they are portable due to their miniaturizability and fast speed analysis (minutes to hours), and are relatively easy to interpret. A total of 2011 articles were found, of which 86 original papers were subsequently evaluated (the majority of which are focused on human pathogens, whereas articles dealing with plant pathogens are in the minority). Thirty-two species of viruses were included in the evaluation. It was found that most of the examined research studies (77%) used nanotechnological modifications. Other ones performed immunological (52%) or genetic analyses (43%) for virus detection. 5% of the reports used peptides to increase the method’s sensitivity. When evaluable, 65% of the research studies had LOD values in the order of ng or nM. The vast majority (79%) of the studies represent proof of concept and possibilities with low application potential and a high need of further research experimental work.
Ligand-receptor interactions (LRIs) are the basis for all the biological processes taking place in living cells and have been exploited to develop and implement in medical field a number of highly sensitive biosensors for the detection of various biomarkers in complex biological fluids. Drug-target interactions, one of the LRIs, are important to understand the biological processes that further help in developing new and better therapeutic molecules. Biosensors based on these interactions give us an idea for the need of modification of existing drugs or to develop new drugs. Common approach to develop biosensors requires the labeling; however, label-free systems provide advantages in avoiding the chances of conformational changes, off-site labeling, and labeling-based hindrances, thus saving time and effort toward assay development. Preliminary drug screening assays are carried out in two-dimensional (2D) models, followed by animal models, which require huge capital investment to reach from bench-top to clinical trials, where only 21% of new compounds make way to phase-1 clinical trials. Three-dimensional culture or organoid culture or organ-on-chip technology has made way for predictive and complex in vitro approach that recapitulates human physiology and represents more similar in vivo behavior than 2D. Multiplexing and nanotechnology have remarkably enhanced the efficacy of biosensors and might lead to a generation of miniaturized biosensors and more than just point-of-care kits. This review provides in-depth analysis of different types of biosensor assays based on drug-target interactions, their advantages, and limitations based on cost, sensitivity, and selectivity and industrial applications.
We propose catalytically synthesized nanozymes based on Prussian Blue (PB) and azidomethyl-substituted poly (3,4-ethylenedioxythiophene) (azidomethyl-PEDOT) as novel electrocatalytic labels for DNA/RNA sensors. Catalytic approach allowed to synthesize highly redox and electrocatalytically active Prussian Blue nanoparticles functionalized with azide groups that enable 'click' conjugation with alkyne-modified oligonucleotides. Both competitive and sandwich-type schemes were realized. As the sensor response the direct (mediator-free) electrocatalytic current of H2O2 reduction can be measured, which is proportional to the concentration of the hybridized labeled sequences. The current of H2O2 electrocatalytic reduction is only 3-8 times increased in the presence of the freely diffusing mediator catechol, which indicates high efficiency of direct electrocatalysis with the elaborated labels. Electrocatalytic amplification of the signal allows robust detection of (63-70)-base target sequences with concentrations below 0.2 nM in blood serum within an hour. We believe, the use of advanced Prussian Blue based electrocatalytic labels sets new avenues for point-of-care DNA/RNA sensing.
Nucleic acids are the fundamental building blocks of life and are found in all living things. In recent years, their functions have been shown to extend beyond the Watson-Crick base pair recognition of complementary strands. Molecules (known as aptamers) consisting of 40-50 nucleotides have been isolated that are able to bind a broad range of molecules with high affinity and specificity. The molecules recognized by aptamers range from small organic molecules to proteins, cells and even intact viral particles. Catalytic DNA molecules called NAzymes (RNAzyme or DNAzyme) have also been shown to exist and, when combined with aptamers, are known as aptazymes. These biomolecules can be used to develop smart and innovative biosensors for environmental analysis. Monitoring of contaminants in the air, water and soil is a key component in understanding and managing risks to human health and ecosystems. This, in conjunction with the time and cost involved in traditional chemical analysis, means there is a growing need for simple, rapid, cost-effective and portable screening methods. Biosensors are compact devices which complement current field screening and monitoring methods.
This book demonstrates the incredible opportunities that nucleic acids can offer to environmental analytical chemistry. The chapters: show how nucleic acids have a pivotal role in the development of smart biosensors for environmental monitoring; describe the development of biosensors based on aptamers and NAzymes for the detection of organic and inorganic pollutants; deal with the use of nucleic acid based biosensors for environmental toxicity screening, and detail the use of nanomaterials, as well as miniaturization and lab-on-a-chip technologies, for nucleic acid based biosensing systems.
In this Opinion, redox transformations of bioactive electron donors and electron acceptors, especially reactive electrophiles, are discussed. The mutual balance and their cooperative effects, together with an extensive battery of antioxidant/prooxidant enzymes, is responsible for maintaining the redox homeostasis of the cell. Electrochemical approaches play a key role in the investigation of these molecules, especially in research of their physico-chemical properties, their stability/reactivity and ability to interact with other cellular components such as nucleic acids, proteins (enzymes and cell receptors) and lipid membranes. The interaction of electron acceptors/donors is the basis for an understanding of highly complex and dynamic systems for homeostasis control and the cell response to stress conditions.
Electrochemical-sensing interfaces have relied on carbon’s unique electrochemical/bio/gas sensors characteristics for a long time. Furthermore, recent advancements in material design and synthesis, particularly nanomaterials, have resulted in very reliable electrochemical sensing devices with better analytical performance. Carbon nanoparticles’ extraordinary sensitivity to changes in surface conductivity caused by the presence of adsorbates allows them to be used as very sensitive. Carbon nanomaterial-based modified electrodes have also proven their capacity to serve as anchors for biomolecules, like nucleic acids, DNA, and to reduce surface fouling. As a result, carbon nanoparticles are attracting a lot of attention from researchers as a foundation for a lot of electrochemical sensors. Similarly, the electrochemical/bio/gas sensor characteristics of synthetic diamonds, such as high chemical inertness and biocompatibility, make it suitable for biochemical sensing as well as the electrochemical interface for biological systems. The recent development of carbon nanomaterial-based sensors and associated interfaces exemplifies this concept.
As a kind of excellent biomaterials, deoxyribonucleic acids (DNAs) have been employed to build a variety of biosensors via the interactions between DNAs and biomolecules or chemical compounds. The DNA-based electrochemical biosensors with the advantages of easy operation, rapid response and low cost have been widely used in biochemical analysis with high sensitivity and selectivity. However, most of the DNA-based electrochemical biosensors require the labeling of electroactive molecules or nanomaterials on DNAs as signal read-out elements, which thus inevitably suffer from complicated operation and expensive cost. Label-free strategies are alternatives for DNA-based electrochemical biosensors without additional assay reagents and tedious procedures. Because of the merits of simplicity and low cost, DNA-based label-free electrochemical biosensors have attracted tremendous attention from researchers as a promising analytical technology. In this review, the principles and applications of DNA-based label-free electrochemical biosensing systems including heterogeneous and homogeneous modes with various amplification strategies have been introduced and summarized systematically. Furthermore, the present challenges and further perspectives of the DNA-based label-free electrochemical biosensors are highlighted.
An interdisciplinary science, such as physics, material science, biochemistry, and biotechnology, microbiology environmental chemistry has attracted graphene in the synthesis and application due to features such as surface to volume ratio, strength to weight ratio, defects over these surfaces, fine tunable properties such as dimensions, lengths, and twisted form. The option is wide open due to sp² carbon for the creation for multidimensional characteristic properties such as electronic, presence of optical features.
The bipolar and nano nature in graphene synthesis methodology creates a conducible environment for biomolecules such as enzymes, nucleic acids, which in due course are responsible for the development of sensors for hydrogen peroxide, antioxidants, pharmaceutically important drugs and ammonia. Furthermore the commercialization has scaled up production in the industrial sector. Based on the extensive literature survey, reviewer intends to bring out some research related to graphene synthesis chemistry, physico and electrochemical properties, biotechnical functionalization and sensing technology.
This laboratory experiment introduces the fundamentals of electrochemical techniques to the undergraduate students by developing a simple and cost-effective miniaturized electrochemical setup for detecting UV-induced DNA damage. This is an imperative experiment, as it provides students first-hand experience with electrochemical techniques while reinforcing lecture material on the biosensing applications. In this experiment, a simple and daily use material such as an ordinary pencil lead acts as a platform for an electrochemical DNA sensor. A three-electrode cell setup is utilized to monitor UV-induced DNA damage. Students will test the hypothesis about UV-induced DNA damage by measuring the redox signal of guanine under various experimental conditions. The simple, fast, and inexpensive setup allows students to examine the factors that would influence the electrochemical signals. This experimental work provides an opportunity for students to learn how to design a simple electrochemical experiment for research purposes as well as handling the biological samples. Proposing a hypothesis and testing it by designing an experiment are the experiences that will be gained by the students while learning about the optimization and validation of an analytical technique.
Development of sensitive, rapid and low-cost DNA detection tools and elucidation of the structural changes of DNA after interaction with chemotherapeutic drugs is of great significance. In this respect, the objective of this work was to construct a DNA biosensor based on electrodeposited cetyl trimethylammonium bromide-multiwalled carbon nanotubes (poly(CTAB-MWCNTs)) composite on single-use graphite electrodes (PGEs) for the direct detection of dsDNA and further implementation for the anticancer drug screening. The morphology and electrochemical characterization of the fabricated biosensor were utilized. The new surfactant-carbon nanotubes based electrode with porous composite structure led to significant improvement for the detection of dsDNA in the linear range of 4–150 μg mL⁻¹ with detection limit of (LOD) 3.06 μg mL⁻¹. Under the optimized conditions, the dsDNA modified poly(CTAB-MWCNTs)/PGEs were utilized to evaluate the binding interaction of an anticancer drug, irinotecan (CPT-11) with DNA by differential pulse voltammetry (DPV) and also by UV-vis spectroscopy. The guanine oxidation signal was linear in a CPT-11 concentration range of 2−500 μg mL⁻¹ with LOD of 1.03 μg mL⁻¹. Moreover, equilibrium constant (K) for the binding process was determined as 6.84x10⁴ M⁻¹. The applicability of the was examined by quantitative measuring of CPT-11 in serum samples with high recoveries (106.0−98.2%). The proposed DNA biosensor hold considerable promise for different biointeraction applications.
A simple and easy-operation electrode modification strategy was proposed using Cu-MOF/GO nanohybid for physiologists and pathologists to the feasible and reliable simultaneous electrochemical detections of DNA bases, guanine and adenine. The nanohybid was prepared by simple ultrasonic method and was employed for fabrication of sensing interface. SEM, TEM, XRD, FT-IR and electrochemical characterizations were used to characterize general morphology and the structure of the nonohybrid. The proposed Cu-MOF/ERGO/GCE shows ultra-stable and high-sensitivity performance in simultaneous electrochemical detection of guanine and adenine. The recorded DPV curves reveal a linear increase in faradic signals with increases in concentrations of guanine and adenine in the range of 0.02−10 µM and 20−100 µM for guanine, and 0.005−20 µM and 40−200 µM for adenine.The relative standard deviation of guanine and adenine for 50 consecutive detection is 1.37% and 1.92%, respectively. It was proved that the proposed Cu-MOF/ERGO/GCE can be performed to the detection of guanine and adenine in real samples, Herring sperm DNA, and satisfactory results were obtained. This strategy does not require complicated modification procedures, professional modification techniques, or sophisticated instruments, but it can provide a highly sensitive and stable detection method, which is expected to expand and deepen the application of electrochemical detection in life science research.
Using a series of specifically designed oligonucleotides we have identified adenine and cytosine nucleobases as residues involved in catalytic hydrogen evolution reaction (CHER) of nucleic acids at the hanging mercury drop electrode (HMDE). Due to the CHER, nucleic acids yield catalytic peak HNA allowing their label-free and reagent-less analysis at low concentrations. Additionally, our results suggest that presence of the electroactive bases (adenine and cytosine) facilitates guanine reduction which is for the first time linked to a signal measurable at the HMDE — the peak P. We assume that the peak P is connected with reduction of guanine to 7,8-dihydroguanine, of which reoxidation to guanine is detected at the electrode by the earlier described anodic peak G. Processes connected with the above mentioned signals were studied using cyclic voltammetry by inspection of dependences on experimental parameters such as negative vertex potential and pH.
Mercury is a truly unique electrode material, which due to its liquid state offers significant advantages, not accessible to solid electrodes. With normal, good laboratory procedures it has been and can be handled safely. It has broad range of applications in physical electrochemistry and electroanalytical chemistry. In this article three somewhat unusual examples of application of mercury electrode are presented: its use as a liquid junction-free reference electrode, as a miniature Langmuir trough for scanning tunneling microscopy and as a new tool for electrochemistry of biomacromolecules.
Recently we have demonstrated that pyrolytic graphite electrodes (PGE) offer a broad analytically useful potential window, ranging from about −2.0 V to +1.6 V vs. Ag|AgCl|3 M KCl, which enables to use the PGE not only for anodic measurements, but also for direct reduction of nucleobases in DNA oligonucleotides. In this follow‐up study, we have focused on the electrochemical behavior of four 2′‐deoxynucleosides derived from adenine, guanine, cytosine, thymine, uracil, and 5‐methyl cytosine on the PGE. On one hand we have obtained analogous primary redox responses as previously with the oligonucleotides. On the other hand, significant differences were observed, particularly when considering secondary responses involving products of the primary conversions, suggesting involvement of different mechanisms. Further we have found that presence of the ambient oxygen in the electrolyte does not dramatically affect the redox signals of the nucleosides. This finding is in contrast with DNA responses measured at the mercury‐based electrodes, where deaeration prior to the measurements was necessary. We demonstrate that all studied nucleosides can be analyzed using a simple ex situ (medium exchange) procedure.
Grâce aux technologies de la microfluidique (i.e. la manipulation d'un fluide dans un système ayant une dimension caractéristique sub-millimétrique), il est possible d'imaginer l'intégration de l'ensemble des fonctions ordinairement réalisées en laboratoire dans un système miniaturisé, réalisant ainsi un laboratoire sur puce. Cela peut ainsi permettre d'allier efficacité et bas-coût requis pour la réalisation de dispositif de diagnositcs médicaux utilisable en dehors d'infrastructure médicalisée, souvent appelés systèmes Point-of-Care. Pour la réalisation d'un tel dispositif, il semble important de concevoir l'intégration des différents composants du système d'une façon cohérente, et en prenant en compte l'ensemble des contraintes imposées par l'application finale ciblée. Le travail effectué au cours de cette thèse a ainsi été réalisé dans l'optique de proposer une réponse à cette problématique d'intégration dans le cadre du développement d'un système microfluidique de diagnostic Point-of-Care basé sur une réaction d'amplification d'ADN isotherme LAMP. Afin de pouvoir proposer un système bon marché et dont l'industrialisation est aisée, nous avons fait appel à l'utilisation du papier comme support et au thermoformage comme moyen de production. En effet à la fois l'industrie papetière et le procédé de thermoformage sont d'ores et déjà existant et proposent des fabrications en série. De plus, le faible coût du matériau et du procédé en question permettent d'envisager un dispositif final à bas-coût. Afin de pouvoir effectuer et détecter la réaction de LAMP la présence de fonctions actives telles qu'un chauffage et un outil de détection est nécessaire. Pour ces dernières, l'intégration a été réalisée par procédé sérigraphique. Le chauffage est effectué par effet Joule grâce au dépôt d'une couche d'encre conductrice à base de carbone. La détection est quant à elle faite par méthode potentiométrique, à l'aide d'électrode couverte de polyaniline. Il sera également montré que l'utilisation de ces méthodes de fabrication est pertinente en termes d'intégration car elles permettent une superposition des différentes fonctions actives, mais également leur intégration directement dans le système microfluidique.
In 1941 J. Heyrovský invented Oscillographic Polarography (OP) with controlled alternating current (a.c.). The method was developed during the World War and the first commercially available instruments for OP were produced in Czechoslovakia at the beginning of the 1950’s. Such instruments offered derivative and cyclic OP curves allowing simple analysis of various compounds and stimulated the publication of hundreds of papers. Thanks to OP, electrochemical analysis of DNA started already in 1958. According to the present nomenclature, OP can be denominated as a.c. chronopotentiometry. Surprisingly, authors who later described derivative and cyclic chronopotentiometry did not consider J. Heyrovský’s OP methodology in their work. Present electrochemical studies of biomacromolecules take advantage of modern instruments for constant current chronopotentiometric stripping, allowing DNA and protein structure-sensitive analysis. Recent progress in these studies is reviewed.
Recent progress in label-free electrochemical analysis of biomacromolecules, such as proteins, nucleic acids and carbohydrates is reviewed. Since the 1970s electrochemical analysis of proteins focused on non-protein redox-active components of a relatively small group of conjugated proteins. In the recent decade, the ability of practically of all proteins to catalyze hydrogen evolution at mercury-containing electrodes was utilized for development of the protein structure-sensitive analysis. Some amino acid residues, such as arginine, lysine and cysteine contribute to the catalytic hydrogen evolution reaction (CHER) at neutral pH yielding protein reduction signals at highly negative potentials. It was found that native proteins do not lose their folded structure when adsorbed at mercury electrode close to the potential of zero charge. Surface-attached proteins get however denatured due to the electric field effects during their prolonged exposure to negative potentials. Using the constant current chronopotentiometric stripping it was possible to limit the exposure time to milliseconds preventing protein denaturation. The method was utilized in detection of changes in protein structures due to mutation, chemical modification, aggregation, damage by environmental agents, as well as to studies of poorly soluble membrane proteins, DNA–protein and protein–protein interactions, etc. Application of voltammetric methods, such as fast scan CV and normal pulse voltammetry showed smaller sensitivity to tiny changes in protein structures. Recently CHER was found also in some polysaccharides such as chitosan and in NH2 group-containing glycans. Very recent development in electrochemical analysis of DNA and RNA was briefly summarized.
We show for the first time that DNA produces an electrocatalytic square wave voltammetric reduction peak or chronopotentiometric peak HDNA at mercury and solid amalgam electrodes. These peaks are due to the catalytic hydrogen evolution reaction, allowing label-free detection of chromosomal DNA and oligodeoxynucleotides (ODNs) below ppm and at subnanomolar levels, respectively. Using peak HDNA at high current densities, native chromosomal DNA was distinguished from its denatured form. With this method, damage of DNA from human cancer cells by ionizing radiation or sonication was detected. Sensitivity of the DNA determination was greatly enhanced in the presence of [Co(NH3)6]3+. Chromosomal DNA and ODNs can be detected at concentrations by 2-3 orders lower as compared to any earlier voltammetric or chronopotentiometric label-free methods. In the presence of 4 mM [Co(NH3)6]3+, DNA from human cancer cells was easily detected at 5 ng/mL, i.e., below the concentration level of cell-free DNA in body fluids.
Analysis by means of oscillographic polarography with alternating current is discussed and illustrated by luminous diagrams showing tlie dependence —V. The quantity of the depolarizer can be determined either by the area of the cut-in on the diagram or by a comparative titration method.Practical examples are given in oscillographic analysis of various sulphonamides, local anaesthetics, purine derivatives, alkaloids, pyridin carboxylic acids, vitamins, antibiotics, hormones, etc.RésuméL'analyse par polarographie oscillographique, en courant altcrnatif, est discutée et illustrée de photographies, (diagrammes obtenus sur l'oscilloscope) démontrant la relation — V. La quantité de dépolarisant peut être déterminée, soit par la surface de l'encoche, soit par une méthode de titration comparative. Des exemples pratiques sont donnés: analyse oscillographique divers sulfamidés d'anesthésiques locaux, de dérivés de la purine, d'alcaloides, d'acides pyridine-carboxyhques, de vitamines, d'antibiotiques, d'hormones, etc.ZusammenfassungDie oszillographisch-polarographische Analyse mit Wechselstrom wird besprochen und die Abhängigkeit —V durch veranschaulichende Leuchtdiagramme illustriert. Die Quantität des Depolarisators kann entweder aus der Fläche des Einschnittes am Diagranim oder durch eine vergleichende Titrationsmethode ermittelt werden.Es werden praktische Beispiele der oszillographischen Analyse verschiedener Sulfonamide, Lokalanästhetika, Purinderivate, Alkaloide, Pyridincarboxylsäuren, Vitamine, Antibiotika, Hormone, usw. angeführt.