Aptamer and Electrochemical Aptasensor towards Selenate Ions (SeO42-).

Selenium is an essential inorganic compound in human and animal nutrition, involved in the proper functioning of the body. As a micronutrient, it actively contributes to the regulation of various metabolic activities, i.e., thyroid hormone, and protection against oxidative stress. However, Se exhibits a narrow concentration window between having a positive effect and exerting a toxic effect. In higher doses, it negatively affects living organisms and causes DNA damage through the formation of free radicals. Increased reactivity of Se anions can also disrupt the integrity and function of DNA-repairing proteins. As the permissible concentration of Se in drinking water is 10 µg/L, it is vital to develop sensitive and robust methods of Se detection in aqueous samples. In this study, for the first time, we proposed a selective aptamer for selenate ion detection, chosen following the SELEX process, and its application in the construction of an electrochemical aptasensor towards SeO42- ions. Measurement conditions such as the used redox marker and pH value of the measurement solution were chosen. The proposed aptasensor is characterized by good selectivity and an LOD of 1 nM. Conditions for biosensor regeneration and storage were also investigated in this research.

Identification of medium- and mechanism-related pitfalls towards improved performance and applicability of electrochemical mercury(II) aptasensors.

The importance of understanding the mercury (II) ion interactions with thymine-rich DNA sequences is the reason for multiple comparative investigations carried out with the use of optical detection techniques directly in the depth of solution. However, the results of such investigations have limited applicability in the interpretation of the Hg2+ binding phenomenon by DNA sequences in thin, interfacial (electrode/solution), self-organized monolayers immobilized on polarizable surfaces, often used for sensing purposes in electrochemical biosensors. Overlooking the careful optimization of the measurement conditions is the source of discrepancies in the interpretation of the registered electrochemical signal. In this study, the chosen effects accompanying the efficiency of surface related recognition of Hg2+ by polyThymine DNA sequences labelled with methylene blue were investigated by voltammetry, QCM and spectro-electrochemical techniques. As was shown, the composition of the biosensing layer and buffers or the analytical procedures have a significant impact on the registered electrochemical readout which translates into signal stability, the biosensor's working parameters or even the mechanism of detection. After elucidation of the above factors, the complete and ready-to-use biosensor-based analytical solution was proposed offering subpicomolar mercury ion determination with high selectivity (also in aqueous real samples), reusability, and high signal stability even after long-term storage. The developed procedures were successfully used during the miniaturization process with self-prepared (PVD) elastic transducers. The obtained sensor, together with the simplicity of its use, low manufacturing cost, and attractive analytical parameters (i.e., LOD < < Hg2+ WHO limit) can present an interesting alternative for on-site mercury ion detection in environmental samples.

Modern Electrochemical Biosensing Based on Nucleic Acids and Carbon Nanomaterials.

To meet the requirements of novel therapies, effective treatments should be supported by diagnostic tools characterized by appropriate analytical and working parameters. These are, in particular, fast and reliable responses that are proportional to analyte concentration, with low detection limits, high selectivity, cost-efficient construction, and portability, allowing for the development of point-of-care devices. Biosensors using nucleic acids as receptors has turned out to be an effective approach for meeting the abovementioned requirements. Careful design of the receptor layers will allow them to obtain DNA biosensors that are dedicated to almost any analyte, including ions, low and high molecular weight compounds, nucleic acids, proteins, and even whole cells. The impulse for the application of carbon nanomaterials in electrochemical DNA biosensors is rooted in the possibility to further influence their analytical parameters and adjust them to the chosen analysis. Such nanomaterials enable the lowering of the detection limit, the extension of the biosensor linear response, or the increase in selectivity. This is possible thanks to their high conductivity, large surface-to-area ratio, ease of chemical modification, and introduction of other nanomaterials, such as nanoparticles, into the carbon structures. This review discusses the recent advances on the design and application of carbon nanomaterials in electrochemical DNA biosensors that are dedicated especially to modern medical diagnostics.

Studies on the application of single-stranded DNA and PNA probes for electrochemical detection of miRNA 141.

The abnormal concentration of microRNAs (miRNAs) can be associated with occurrence of various diseases including cancer, cardiovascular and neurodegenerative, hence they can be considered as potential biomarkers. An attractive approach could be the application of electrochemical methods, particularly where hybridization event between single-stranded deoxyribonucleic acid (ssDNA) or peptide-nucleic acid (PNA) with miRNA strand happens. Recently, the use of various nanomaterials such as gold nanoparticles, graphene oxide, quantum dots as well as catalyzed hairpin assembly or hybridization chain reaction were proposed to further enhance the performance of elaborated sensors. Herein, we present the studies on selection of receptor layer composition for detection of miRNA 141. The possibility of formation of receptor layer and further duplex monolayer between ssDNA or PNA with miRNA was analyzed by atomic force microscopy (AFM) technique. The interaction of ssDNA and PNA probes with miRNA was further verified using surface plasmon resonance (SPR) and quartz - crystal microbalance (QCM) techniques. On the basis of impedance spectroscopy it was shown that the use of unlabelled ssDNA as receptor layer provided 0.1 pM detection limit. This shows that proposed biosensor that is simple in preparation and use is an attractive alternative to other recently presented approaches.

Recent Achievements in Electrochemical and Optical Nucleic Acids Based Detection of Metal Ions.

Recently nucleic acids gained considerable attention as selective receptors of metal ions. This is because of the possibility of adjusting their sequences in new aptamers selection, as well as the convenience of elaborating new detection mechanisms. Such a flexibility allows for easy utilization of newly emerging nanomaterials for the development of detection devices. This, in turn, can significantly increase, e.g., analytical signal intensity, both optical and electrochemical, and the same can allow for obtaining exceptionally low detection limits and fast biosensor responses. All these properties, together with low power consumption, make nucleic acids biosensors perfect candidates as detection elements of fully automatic portable microfluidic devices. This review provides current progress in nucleic acids application in monitoring environmentally and clinically important metal ions in the electrochemical or optical manner. In addition, several examples of such biosensor applications in portable microfluidic devices are shown.

Detection of SARS-CoV-2 Using Reverse Transcription Helicase Dependent Amplification and Reverse Transcription Loop-Mediated Amplification Combined with Lateral Flow Assay.

Rapid and accurate detection and identification of pathogens in clinical samples is essential for all infection diseases. However, in the case of epidemics, it plays a key role not only in the implementation of effective therapy but also in limiting the spread of the epidemic. In this study, we present the application of two nucleic acid isothermal amplification methods-reverse transcription helicase dependent amplification (RT-HDA) and reverse transcription loop-mediated amplification (RT-LAMP)-combined with lateral flow assay as the tools for the rapid detection of SARS-CoV-2, the etiological agent of COVID-19, which caused the ongoing global pandemic. In order to optimize the RT-had, the LOD was 3 genome copies per reaction for amplification conducted for 10-20 min, whereas for RT-LAMP, the LOD was 30-300 genome copies per reaction for a reaction conducted for 40 min. No false-positive results were detected for RT-HDA conducted for 10 to 90 min, but false-positive results occurred when RT-LAMP was conducted for longer than 40 min. We concluded that RT-HDA combined with LFA is more sensitive than RT-LAMP, and it is a good alternative for the development of point-of-care tests for SARS-CoV-2 detection as this method is simple, inexpensive, practical, and does not require qualified personnel to perform the test and interpret its results.

Comparative Evaluation of Different Surface Coatings of Fe3O4-Based Magnetic Nano Sorbent for Applications in the Nucleic Acids Extraction.

Nucleic acid extraction and purification are crucial steps in sample preparation for multiple diagnostic procedures. Routine methodologies of DNA isolation require benchtop equipment (e.g., centrifuges) and labor-intensive steps. Magnetic nanoparticles (MNPs) as solid-phase sorbents could simplify this procedure. A wide range of surface coatings employs various molecular interactions between dsDNA and magnetic nano-sorbents. However, a reliable, comparative evaluation of their performance is complex. In this work, selected Fe3O4 modifications, i.e., polyethyleneimine, gold, silica, and graphene derivatives, were comprehensively evaluated for applications in dsDNA extraction. A family of single batch nanoparticles was compared in terms of morphology (STEM), composition (ICP-MS/MS and elemental analysis), surface coating (UV-Vis, TGA, FTIR), and MNP charge (ζ-potential). ICP-MS/MS was also used to unify MNPs concentration allowing a reliable assessment of individual coatings on DNA extraction. Moreover, studies on adsorption medium (monovalent vs. divalent ions) and extraction buffer composition were carried out. As a result, essential relationships between nanoparticle coatings and DNA adsorption efficiencies have been noticed. Fe3O4@PEI MNPs turned out to be the most efficient nano sorbents. The optimized composition of the extraction buffer (medium containing 0.1 mM EDTA) helped avoid problems with Fe3+ stripping, which improved the validity of the spectroscopic determination of DNA recovery.

Molecular diagnostic of toxigenic Corynebacterium diphtheriae strain by DNA sensor potentially suitable for electrochemical point-of-care diagnostic.

The presented study is focused on the development of electrochemical genosensor for detection of tox gene fragment of toxigenic Corynebacterium diphtheriae strain. Together with our previous studies it fulfils the whole procedure for fast and accurate diagnostic of diphtheria at its early stage of infection with the use of electrochemical methods. The developed DNA sensor potentially can be used in more sophisticated portable device. After the electrochemical stem-loop probe structure optimization the conditions for real asymmetric PCR (aPCR) product detection were selected. As was shown it was crucial to optimize the magnesium and organic solvent concentrations in detection buffer. Under optimal conditions it was possible to selectively detect as low as 20.8 nM of complementary stand in 5 min or 0.5 nM in 30 min with sensitivity of 12.81 and 0.24 1⋅µM-1 respectively. The unspecific biosensor response was elucidated with the use of new electrode blocking agent, diethyldithiocarbamate. Its application in electrochemical genosensors lead to significant higher current values and the biosensor response even in conditions with magnesium ion depletion. The developed biosensor selectivity was examined using samples containing genetic material originated from a number of non-target bacterial species which potentially can be present in the human upper respiratory tract.

From Small Molecules Toward Whole Cells Detection: Application of Electrochemical Aptasensors in Modern Medical Diagnostics.

This paper focuses on the current state of art as well as on future trends in electrochemical aptasensors application in medical diagnostics. The origin of aptamers is presented along with the description of the process known as SELEX. This is followed by the description of the broad spectrum of aptamer-based sensors for the electrochemical detection of various diagnostically relevant analytes, including metal cations, abused drugs, neurotransmitters, cancer, cardiac and coagulation biomarkers, circulating tumor cells, and viruses. We described also possible future perspectives of aptasensors development. This concerns (i) the approaches to lowering the detection limit and improvement of the electrochemical aptasensors selectivity by application of the hybrid aptamer-antibody receptor layers and/or nanomaterials; and (ii) electrochemical aptasensors integration with more advanced microfluidic devices as user-friendly medical instruments for medical diagnostic of the future.

Electrochemistry of Graphene Nanoplatelets Printed Electrodes for Cortical Direct Current Stimulation.

Possible risks stemming from the employment of novel, micrometer-thin printed electrodes for direct current neural stimulation are discussed. To assess those risks, electrochemical methods are used, including cyclic voltammetry, square-wave voltammetry, and electrochemical impedance spectroscopy. Experiments were conducted in non-deoxidized phosphate-buffered saline to better emulate living organism conditions. Since preliminary results obtained have shown unexpected oxidation peaks in 0-0.4 V potential range, the source of those was further investigated. Hypothesized redox activity of printing paste components was disproven, supporting further development of proposed fabrication technology of stimulating electrodes. Finally, partial permeability and resulting electrochemical activity of underlying silver-based printed layers of the device were pointed as the source of potential tissue irritation or damage. Employing this information, electrodes with corrected design were investigated, yielding no undesired redox processes.

The influence of a swab type on the results of point-of-care tests.

Most point-of-care tests (POCT) use swabs for sampling and/or for applying a sample on the test. A variety of swabs differing in tip materials is commercially available. Different tip materials have different chemical and physical characteristics which might influence the specimen collection and release. We investigated properties of various types of swabs used in clinical diagnostics with focusing on two kinds of analytes, DNA and proteins, which are most often used targets in POCT. As the model samples we used diphtheria toxoid NIBSC 69/017 for investigating recovery of protein analytes such as antigens and bacterial strains of Escherichia coli ATCC 25922, diphtheria toxin-producing Corynebacterium diphtheriae NCTC 10648, and the clinical isolate nontoxigenic C. diphtheriae 5820/15 for investigating the recovery of nucleic acids. We investigated four types of swabs most commonly used in clinical diagnostics in terms of absorption capacity and efficiency of release of nucleic acids and proteins. Volume uptake was measured in milligrams. For DNA release various washing out buffers were used and the amount of released DNA was measured spectrophotometrically. The amount of protein released from the swabs were examined using the Lowry assay. We observed statistically significant differences (p < 0.05) in the mean weights of absorbed liquid, in the DNA recovery and protein recovery by the four variety of swab examined. However, the efficiency of DNA and protein release was not correlated to the absorbed volume of a sample, but rather to the properties of swabs. The swab composition and structure can have a significant impact on the collection and release efficiency of a sample. Therefore, validation of POCT in relation to the used swabs for sampling is really important. The use of inappropriate swabs could lead to false negative or misleading analysis results.

Reduced nonspecific protein adsorption by application of diethyldithiocarbamate in receptor layer of diphtheria toxoid electrochemical immunosensor.

The immunoassay technology is of particular importance for both the environmental industry and clinical analysis. Biosensors, with the sensing layer based on antibodies or their fragments, offer high selectivity and short detection times. However, analytical devices where the electrochemical signal corresponds to changes in the interfacial region (sensing layer/electrode surface) are very susceptible to any nonspecific adsorption. Unfortunately, proteins (including antibodies) belong to the molecules showing high non-specific interactions with solid substrates. Herein, we propose diethyldithiocarbamate as a new antifouling and highly conductive agent. The investigations were conducted to evaluate its interaction with chosen proteins and the mechanism of its co-adsorption with biotinylated thiol (an anchor point for immune-sensing elements). The developed receptor layer is characterised by reduced nonspecific protein adsorption and high conductivity with the same preserved specificity of the antibodies (immobilised by the streptavidin/biotin bioaffinity technique). This allowed for selective detection of the diphtheria toxoid, an inactive toxin secreted by virulent strains of Corynebacterium diphtheria, at the level of 5 â‹… 10-6 µg⋅ml-1 (1 â‹… 10-6 Lf⋅ml-1) and in the real-life sample.

The electrochemical profiles of Auranofin and Aubipyc, two representative medicinal gold compounds: A comparative study.

A micro-electrochemical reaction cell was coupled to an electrospray mass spectrometer in order to track redox transformations for two representative medicinal gold compounds - i.e. [(2,3,4,6-tetra-O-acetyl-1-thio-ß-D-glucopyranosato-S)(triethylphosphine)gold(I)] and [Au(bipydmb-H)(OH)][PF6] (where bipydmb-H is deprotonated 6-(1,1-dimethylbenzyl)-2,2'-bipyridine), known as Auranofin and Aubipyc respectively - in parallel to square wave voltammetry (SWV) measurements. Irreversible oxidation of thio-glucose tetraacetate was the dominant reaction for the gold(I) compound Auranofin; oxidation was accompanied by hydrolysis leading to progressive deacetylation. Two main active forms were identified for this prodrug: the triethylphosphinegold(I) cation and a gold(I) thioglucose species, with a variable number of acetyl groups. For the gold(III) complex Aubipyc irreversible reduction of the gold(III) center was highlighted, accompanied by a ligand exchange process. The free gold(I) ion is proposed to be the final species that subsequently binds transport proteins in the bloodstream. Molecule specific mass spectrometry determinations provide complementary data to square wave voltammetry helping to understand the nature of the electrochemical conversions of complex or unstable compounds. Finally, it was possible to establish that oxidizing conditions during drug preparation and administration should be avoided in the case of Auranofin; conversely, reduction conditions typical for the blood or the cytosol environment are suitable to obtain the active gold(I) species from the gold(III) complex Aubipyc.

DNA Delivery Systems Based on Peptide-Mimicking Cationic Lipids-The Effect of the Co-Lipid on the Structure and DNA Binding Capacity.

In continuation of previous work, we present a new promising DNA carrier, OO4, a highly effective peptide-mimicking lysine-based cationic lipid. The structural characteristics of the polynucleotide carrier system OO4 mixed with the commonly used co-lipid DOPE and the saturated phospholipid DPPE have been studied in two-dimensional and three-dimensional model systems to understand their influence on the physical-chemical properties. The phase behavior of pure OO4 and its mixtures with DOPE and DPPE was studied at the air-water interface using a Langmuir film balance combined with infrared reflection-absorption spectroscopy. In bulk, the self-assembling structures in the presence and absence of DNA were determined by small-angle and wide-angle X-ray scattering. The amount of adsorbed DNA to cationic lipid bilayers was measured using a quartz crystal microbalance. The choice of the co-lipid has an enormous influence on the structure and capability of binding DNA. DOPE promotes the formation of nonlamellar lipoplexes (cubic and hexagonal structures), whereas DPPE promotes the formation of lamellar lipoplexes. The correlation of the observed structures with the transfection efficiency and serum stability indicates that OO4/DOPE 1:3 lipoplexes with a DNA-containing cubic phase encapsulated in multilamellar structures seem to be most promising.

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents.

The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.

Electrochemical oligonucleotide-based biosensor for the determination of lead ion.

The possibility of utilization of gold electrodes modified with short guanine-rich ssDNA probes for determination of Pb(2+) was examined. Interaction between guanine residues and lead ion followed by formation of G-quadruplex structures was confirmed by electrochemical impedance spectroscopy investigations. An external cationic redox label, methylene blue, was employed in voltammetric measurements for analytical signal generation. It was shown that due to the G-quadruplex formation, the oligonucleotides in the recognition layer fold, which enhances the electron transfer between methylene blue and the electrode surface. The MB current signal rises proportionally to the lead ion concentration in the range from 0.05 to 1µmol/L. The developed biosensor demonstrated high selectivity towards Pb(2+) ion, with only minor response towards interfering metal cations. The calculated limit of detection was of 34.7nmol/L. The utilization of the biosensor for Pb(2+) determination in real samples of water was also tested.

Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer.

The present study aims at the further development of the uranyl oligonucleotide-based voltammetric biosensor, which takes advantage of strong interaction between UO2(2+) and phosphate DNA backbone. Herein we report the optimization of working parameters of previously elaborated electrochemical DNA biosensor. It is shown that the sensor sensitivity is highly dependent on the oligonucleotide probe length and the incubation time of sensor in a sample solution. Consequently, the highest sensitivity was obtained for 10-nucleotide sequence and 60 min incubation time. The lower detection limit towards uranyl cation for developed biosensor was 30 nM. The influence of mixed monolayers and the possibility of developing a non-calibration device were also investigated. The selectivity of the proposed biosensor was significantly improved via elimination of adenine nucleobases from the DNA probe. Moreover, the regeneration procedure was elaborated and tested to prolong the use of the same biosensor for 4 subsequent determinations of UO2(2+).

Electrochemical uranyl biosensor with DNA oligonucleotides as receptor layer.

The feasibility of using gold electrodes modified with short-chain ssDNA oligonucleotides for determination of uranyl cation is examined. Interaction between UO(2)(2+) and proposed recognition layer was studied by means of voltammetric and quartz crystal microbalance measurements. It was postulated that ssDNA recognition layer functions via strong binding of UO(2)(2+) to phosphate DNA backbone. The methylene blue was used as a redox marker for analytical signal generation. Biosensor response was based on the difference in electrochemical signal before and after subjecting it to sample containing uranyl ion. The lower detection limit of 30 nmol L(-1) for UO(2)(2+) was observed for a sample incubation time of 60 min. Proposed ssDNA-modified electrodes demonstrated good selectivity towards UO(2)(2+) against common metal cations, with only Pb(2+) and Ca(2+) showing considerable interfering effect.

Electrochemical detection of DNA hybridization using metallacarborane unit.

The evaluation of novel electrochemically active label for electrochemical detection of DNA hybridization is presented. Metallacarborane units modified with iron, cobalt or chromium were investigated. The value of redox potential and relatively strong current signal facilitate usage of Fe-carborane as marker covalently attached to the ssDNA. In electrochemical genosensor the sequence complementary to UL55 gene was labeled and used as a target for biosensor device. Interactions were investigated using electrochemical and piezoelectric methods. Obtained results confirm usefulness of the designed label in electrochemical detection of DNA hybridization.

Application of mass fabricated silicon-based gold transducers for amperometric biosensors.

The backside contact, silicon-based transducers with vacuum-deposited gold layer (BSC) are evaluated as the base for electrochemical biosensors construction. Their comparison with commercially available transducers with screen printed gold and traditional gold disc electrode is reported. To determine the advantages and disadvantages of each of gold surfaces mentioned above, the 6-(ferrocenyl)-hexanethiol was used as the indicator. The results revealed the usefulness of BSC chips for the formation of stable self-assembled monolayers (SAMs). After the preliminary analysis, the SAM of thiol-ssDNA was formed on the BSC transducers as recognition layer. The electrochemical analysis in methylene blue solution was carried out after ssDNA immobilization and DNA-DNA hybridization. It is shown that prepared sensors are able to recognize complementary DNA sequence, based on the change in height and the potential shifts of reduction peaks of methylene blue. Obtained results are in full agreement with literature data. The compact size of silicone-based transducers allows to significantly reduce the required volume of tested solutions.