Search

There are more than 50 neurodegenerative disorders, and amyotrophic lateral sclerosis (ALS) is one of the most common disorders that poses diagnostic and treatment challenges. The poly glycine-proline (polyGP) dipeptide repeat is a toxic protein that has been recognized as a pharmacodynamic biomarker of C9orf72-associated (c9+) ALS, a subtype of ALS that originates from genetic mutation. Early detection of polyGP will help healthcare providers start timely gene therapy. Herein, we developed a label-free electrochemical immunoassay for the simple detection of polyGP in unprocessed cerebrospinal fluid (CSF) samples collected from ALS patients in the National ALS Biorepository. For the first time, an electrografted laser-induced graphene (E-LIG) electrode system was employed in a sandwich format to detect polyGP using a label-free electrochemical impedance technique. The results show that the E-LIG-modified surface exhibited high sensitivity and selectivity in buffer and CSF media with limit of detection values of 0.19 and 0.27 ng/mL, respectively. The precision of the calibration model was better in CSF than in the buffer. The E-LIG immunosensor can easily select polyGP targets in the presence of other dipeptide proteins translated from the c9 gene. Further study with CSF samples from ALS patients demonstrated that the label-free E-LIG-based immunosensor not only quantified polyGP in the complex CSF matrix but also distinguished between c9+ and non-c9- ALS patients.

The urban building stock dataset consists of synthetic input and output data for the energy simulation of one million buildings. The dataset consists of four different residential types, namely: terraced, detached, semi-detached, and bungalow. Constructing this buildings dataset requires conversion, categorization, extraction, and analytical processes. The dataset (in .csv) format comprises 19 input parameters, including advanced features such as HVAC system parameters, building fabric (walls, roofs, floors, door, and windows) U-values, and renewable system parameters. The primary output parameter in the dataset is Energy Use Intensity (EUI in kWh/(m 2 *year)), along with Energy Performance Certificate (EPC) labels categorized on an A to G rating scale. Additionally, the dataset contains end-use demand output parameters for heating and lighting, which are crucial output parameters. jEPlus, a parametric tool, is coupled with EnergyPlus and DesignBuilder templates to facilitate physics-based parametric simulations for generating the dataset. The dataset can be a valuable resource for researchers, practitioners, and policymakers seeking to enhance sustainability and efficiency in urban building environments. Furthermore, dataset holds immense potential for future research in the field of building energy analysis and modeling., (© 2024 The Author(s).)

RNA modification, particularly pseudouridine (Ψ), has played an important role in the development of the mRNA-based COVID-19 vaccine. This is because Ψ enhances RNA stability against nuclease activity and decreases the anti-RNA immune response. Ψ also provides structural flexibility to RNA by enhancing base stacking compared with canonical nucleobases. In this report, we demonstrate the first application of pseudouridine-modified RNA as a probe (Ψ-RNA) for label-free nucleic acid biosensing. It is known that MoS 2 has a differential affinity for nucleic acids, which may be translated into a unique electronic signal. Herein, the Ψ-RNA probe interacts with the pristine MoS 2 surface and causes a change in interfacial electrochemical charge transfer in the MoS 2 nanosheets. Compared with an unmodified RNA probe, Ψ-RNA exhibited faster adsorption and higher affinity for MoS 2 . Moreover, Ψ-RNA could bind to complementary RNA and DNA targets with almost equal affinity when engaged with the MoS 2 surface. Ψ-RNA maintained robust interactions with the MoS 2 surface following the hybridization event, perhaps through its extra amino group. The detection sensitivity of the Ψ-RNA/MoS 2 platform was as low as 500 attomoles, while the results also indicate that the probe can distinguish between complementary targets, single mismatches, and non-complementary nucleic acid sequences with statistical significance. This proof-of-concept study shows that the Ψ-RNA probe may solve numerous problems of adsorption-based biosensing platforms due to its stability and structural flexibility.

Overgrowth of short tandem repeat sequences in our genes can cause various neurodegenerative disorders. Such repeat sequences are ideal targets for the label-free electrochemical detection of such potential expansions. However, their length- and sequence-dependent secondary structures may interfere with the interfacial charge transfer of a detection platform, making them complex targets. In addition, the gene contains sporadic repeats that may result in false-positive signals. Therefore, it is necessary to design a platform capable of mitigating these effects and ultimately enhancing the specificity of tandem repeats. Here, we analyzed three different backbones of nucleic acid microprobes [DNA, peptide nucleic acid, and lock-nucleic acid (LNA)] to detect in vitro transcribed RNA carrying CAG repeats, which are associated with Huntington's disease, based on the charge-transfer resistance of the interface. We found that the LNA microprobe can distinguish lengths down to the attomolar concentration level and alleviate the effect of secondary structures and sporadic repeats in the sequence, thus distinguishing the "tandem repeats" specifically. Additionally, the control experiments conducted with and without Mg 2+ demonstrated the LNA microprobe to perform better in the presence of the divalent cation. The results suggest that the LNA-based platform may eventually lead to the development of a reliable and straightforward biosensor for genetic neurodegenerative disorders.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, which has complex diagnostic steps. Electrochemical immunoassays may make the diagnosis simpler and faster. Here, we present the detection of ALS-associated neurofilament light chain (Nf-L) protein through an electrochemical impedance immunoassay on reduced graphene oxide (rGO) screen-printed electrodes. The immunoassay was developed in two different media, i.e., buffer and human serum, to compare the effect of the media on their figures of merit and calibration models. The label-free charge transfer resistance (R CT ) of the immunoplatform was used as a signal response to develop the calibration models. We found that exposure of the biorecognition layer to human serum improved the impedance response of the biorecognition element with significantly lower relative error. Moreover, the calibration model obtained in the human serum environment has higher sensitivity and a better limit of detection (0.087 ng/mL) than the buffer medium (0.39 ng/mL). The analyses of the ALS patient samples show that concentrations obtained from the buffer-based regression model was higher than the serum-based model. However, a high Pearson correlation (r = 1.00) between the media suggests that concentration in one medium may be useful to predict the concentration in the other medium. Moreover, the Nf-L concentration appears to increase with age in both male and female groups, while overall higher Nf-L was found in the male group than the female group.

We present a PNA microprobe sensing platform to detect trinucleotide repeat mutation by electrochemical impedance spectroscopy. The microprobe platform discriminated Huntington's disease-associated CAG repeats in cell-derived total RNA with S/N 1 : 3. This sensitive, label-free, and PCR-free detection strategy may be employed in the future to develop biosensing platforms for the detection of a plethora of repeat expansion disorders., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

DNA interfaces with nano, micro, and macro materials have gained widespread attention for various applications. Such interfaces exhibit distinct functions and properties not only due to the unique properties of interfacing materials but also sequence- and conformation-dependent characteristics of the DNA. Therefore, DNA interfaces with diverse dimensional materials have advanced our understanding of the interaction mechanisms and the properties of such interfaces. The unique interfacial properties of such novel materials have applications in nanotechnology, biophysics, cell biology, biosensing, and bioelectronics. The field is growing rapidly with the frequent emergence of new interfaces carrying remarkable interfacial character. In this review article, we have classified the DNA interfaces into 0D, 1D, 2D, and 3D categories based on the types of dimensional materials. We review the key efforts made in the last five years and focus on types of interfaces, interfacing mechanisms, and their state-of-the-art applications. This review will draw a general interest because of the diversity in the DNA materials science but also the unique applications that will play a cutting-edge role in biomedical and biosensing research., Competing Interests: Authors claim no conflict of financial or intellectual conflict of interest., (This journal is © The Royal Society of Chemistry.)

DNA is strongly adsorbed on oxidized graphene surfaces in the presence of divalent cations. Here, we studied the effect of DNA adsorption on electrochemical charge transfer at few-layered, oxygen-functionalized graphene (GO x ) electrodes. DNA adsorption on the inkjet-printed GO x electrodes caused amplified current response from ferro/ferricyanide redox probe at concentration range 1 aM-10 nM in differential pulse voltammetry. We studied a number of variables that may affect the current response of the interface: sequence type, conformation, concentration, length, and ionic strength. Later, we showed a proof-of-concept DNA biosensing application, which is free from chemical immobilization of the probe and sensitive at attomolar concentration regime. We propose that GO x electrodes promise a low-cost solution to fabricate a highly sensitive platform for label-free and chemisorption-free DNA biosensing.

Objectives: To our knowledge, this study is the first in the United Arab Emirates (UAE) to investigate the prevalence of child maltreatment in relation to depressive symptoms and self-esteem., Study Design: Exposure to physical maltreatment, emotional abuse and neglect was evaluated in 518 adolescents (86% response rate) randomly selected from schools in Al Ain in the Emirate of Abu Dhabi. The Rosenberg self-esteem scale and the Beck Depression Inventory were used to measure self-esteem and depressive symptoms by using multivariate logistic regression analyses., Results: The mean age of study participants was 14.3 years. Emotional abuse was the most frequent form of maltreatment (33.9%), physical abuse (12.6%) and neglect (12.1%) followed. Male sex was a positive predictor of physical abuse (OR = 2.12; 95% CI 1.18-3.77), whilst higher maternal level of education was protective (OR = 0.40; 95% CI 0.19-0.86). Daily screen time (OR = 2.77; 95% CI 1.17-6.56) and tobacco smoking (OR = 1.86; 95% CI 1.09-3.18) positively predicted emotional abuse. Emotionally maltreated and neglected participants were less likely to report high level of self-esteem and more likely to report symptoms of depression., Conclusions: Child maltreatment in the UAE is of a similar magnitude to what reported in other countries around the world and significantly associated with low self-esteem and depressive symptoms.

Flexible and ultrasensitive biosensing platforms capable of detecting a large number of trinucleotide repeats (TNRs) are crucial for future technology development needed to combat a variety of genetic disorders. For example, trinucleotide CGG repeat expansions in the FMR1 gene can cause Fragile X syndrome (FXS) and Fragile X-associated tremor/ataxia syndrome (FXTAS). Current state-of-the-art technologies to detect repeat sequences are expensive, while relying on complicated procedures, and prone to false negatives. We reasoned that two-dimensional (2D) molybdenum sulfide (MoS 2 ) surfaces may be useful for label-free electrochemical detection of CGG repeats due to its high affinity for guanine bases. Here, we developed a low-cost and sensitive wax-on-plastic electrochemical sensor using 2D MoS 2 ink for the detection of CGG repeats. The ink containing few-layered MoS 2 nanosheets was prepared and characterized using optical, electrical, electrochemical, and electron microscopic methods. The devices were characterized by electron microscopic and electrochemical methods. Repetitive CGG DNA was adsorbed on a MoS 2 surface in a high cationic strength environment and the electrocatalytic current of the CGG/MoS 2 interface was recorded using a soluble Fe(CN) 6 -3/-4 redox probe by differential pulse voltammetry (DPV). The dynamic range for the detection of prehybridized duplexes ranged from 1 aM to 100 nM with a 3.0 aM limit of detection. A detection range of 100 fM to 1 nM was recorded for surface hybridization events. Using this method, we were able to observe selectivity of MoS 2 for CGG repeats and distinguish nonpathogenic from disease-associated repeat lengths. The detection of CGG repeat sequences on inkjet printable 2D MoS 2 surfaces is a forward step toward developing chip-based rapid and label-free sensors for the detection of repeat expansion sequences.

Trinucleotide repeat (TNR) sequences widely exist in nature and their overgrowth is associated with two dozen neurodegenerative diseases in humans. These sequences have a unique helical flexibility, which affects their biophysical properties. A number of biophysical properties of these sequences have been studied in the past except their surface-tethered monolayers. To address the effect of sequence context and the associated helical flexibility on TNR monolayers, disease-relevant TNRs from three flexibility groups were surface-assembled on gold surfaces. The properties of the TNR films were studied, including charge transfer resistance (Rct) by electrochemical impedance spectroscopy (EIS), surface density by chronocoulometry (CC), surface topography by atomic force microscopy (AFM), and electrical conductivity by conducting atomic force microscopy (C-AFM). We found that the TNR film properties are characteristically sequence dependent rather than being dependent on their flexibility rank reported in the literature. The characteristic properties of TNR films studied here may be used for engineering label-free biosensors to detect neurological disorders and build DNA bioelectronics.

Trinucleotide repeat (TNR) sequences introduce sequence-directed flexibility in the genomic makeup of all living species leading to unique non-canonical structure formation. In humans, the expansions of TNR sequences are responsible for almost 24 neurodegenerative and neuromuscular diseases because their unique structures disrupt cell functions. The biophysical studies of these sequences affect their electrophoretic mobility and spectroscopic signatures. Here, we demonstrate a novel strategy to characterize and discriminate the TNR sequences by monitoring their capillary flow in the absence of an external driving force using wax-on-plastic microchannels. The wax-on-plastic microfluidic system translates the sequence-directed flexibility of TNR into differential flow dynamics. Several variables were used to characterize sequences including concentration, single- vs. double-stranded samples, type of repeat sequence, length of the repeat sequence, presence of mismatches in duplex, and presence of metal ion. All these variables were found to influence the flow velocities of TNR sequences as these factors directly affect the structural flexibility of TNR at the molecular level. An overall trend was observed as the higher flexibility in the TNR structure leads to lower capillary flow. After testing samples derived from relevant cells harboring expanded TNR sequences, it is concluded that this approach may transform into a reagent-free and pump-free biosensing platform to detect microsatellite expansion diseases.

Flexible microfluidic devices are currently in demand because they can be mass-produced in resource-limited settings using simple and inexpensive fabrication tools. Finding new ways to fabricate microfluidic platforms on flexible substrates has been a hot area. Integration of customized detection tools for different lab-on-chip applications has made this area challenging. Significant advancements have occurred in the area over the last decade; therefore, there is a need to review such interesting fabrication tools employed on flexible substrates, such as paper and plastics. In this short review, we review individual fabrication tools and their combinations that have been used to develop such platforms in the past five years. These tools are not only simple and low-cost but also require minimal skills for their operation. Moreover, key examples of plastic-based flexible substrates are also presented, because a diverse range of plastic materials have prevailed recently for a variety of lab-on-chip applications. This review should attract audience of various levels, i.e., from hobbyists to scientists, and from high school students to postdoctoral researchers, to produce their own flexible devices in their own settings., Competing Interests: The authors declare no conflict of interest.

DNA repeat expansion sequences cause a myriad of neurological diseases when they expand beyond a critical threshold. Previous electrochemical approaches focused on the detection of trinucleotide repeats (CAG, CGG, and GAA) and relied on labeling of the probe and/or target strands or enzyme-linked assays. However, detection of expanded GC-rich sequences is challenging because they are prone to forming secondary structures such as cruciforms and quadruplexes. Here, we present label-free detection of hexanucleotide GGGGCC repeat sequences, which cause the leading genetic form of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The approach relies on capturing targets by surface-bound oligonucleotide probes with a different number of complementary repeats, which proportionately translates the length of the target strands into charge transfer resistance (R CT ) signal measured by electrochemical impedance spectroscopy. The probe carrying three tandem repeats transduces the number of repeats into R CT with a 3× higher calibration sensitivity and detection limit. Chronocoulometric measurements show a decrease in surface density with increasing repeat length, which is opposite of the impedance trend. This implies that the length of the target itself can contribute to amplification of the impedance signal independent of the surface density. Moreover, the probe can distinguish between a control and patient sequences while remaining insensitive to non-specific Huntington's disease (CAG) repeats in the presence of a complementary target. This label-free strategy might be applied to detect the length of other neurodegenerative repeat sequences using short probes with a few complementary repeats. Graphical abstract Short oligomeric probes with multiple complementary repeats detect long neurodegenerative targets with high sensitivity and transduce into higher impedance signal.

Fabrication of inexpensive and flexible electronic and electrochemical sensors is in high demand for a wide range of biochemical and biomedical applications. We explore hand fabrication of CNT modified AgNPs electrodes using wax-on-plastic platforms and their application in electrochemical immunosensing. Wax patterns were printed on polyethylene terephthalate-based substrates to laydown templates for the electrodes. Hand painting was employed to fabricate a silver conductive layer using AgNPs ink applied in the hydrophilic regions of the substrate surrounded by wax. CNT was drop cast on top of the working electrodes to improve their electrochemical signal. The device layers were characterized by scanning electron microscopy. The electrochemical performance of the hand fabricated AgNPs and CNT/AgNPs electrodes was tested using cyclic voltammetry, differential pulse voltammetry, and amperometry. The electrochemical response of CNT/AgNPs electrodes was relatively faster, higher, and more selective than unmodified AgNPs sensing electrodes. Finally, the hand-painted CNT/AgNPs electrodes were applied to detect carcinoembryonic antigen (CEA) by measuring the end-product of immunoassay performed on magnetic particles. The detection limit for CEA was found to be 0.46 ng/mL.

The first sequence-dependent study of DNA films containing metal-mediated base pairs was performed to investigate the charge transfer resistance (R CT ) of metal-modified DNA. The imidazole (Im) deoxyribonucleoside was chosen as a highly Ag I -specific ligandoside for the formation of Im-Ag I -Im complexes within the duplexes. This new class of site-specifically metal-modified DNA films was characterized by UV, circular dichroism (CD), and X-ray photoelectron spectroscopy (XPS). The electrochemical properties of these systems were investigated by means of electron impedance spectroscopy and scanning electrochemical microscopy. Taken together, these experiments indicated that the incorporation of Ag I ions into the DNA films leads to reduced electron transfer through the DNA films. A simple device was proposed that can be switched reversibly between two distinct states with different charge transfer resistance., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Imaging of toll-like receptor microarrays was achieved using scanning electrochemical microscopy with the successful integration of two ferrocene derivatives in order to enhance the background contrast. This investigation has resulted in the novel fabrication of a tuneable, multiplex, broad-spectrum bacterial sensor for the interrogation of conserved microbial stimuli.

Electrochemiluminescence (ECL) is a sensitive analytical technique with great promise for biological applications, especially when combined with microfluidics. Here, we report the first integration of ECL with digital microfluidics (DMF). ECL detectors were fabricated into the ITO-coated top plates of DMF devices, allowing for the generation of light from electrically excited luminophores in sample droplets. The new system was characterized by making electrochemical and ECL measurements of soluble mixtures of tris(phenanthroline)ruthenium(II) and tripropylamine (TPA) solutions. The system was then validated by application to an oligonucleotide hybridization assay, using magnetic particles bearing 21-mer, deoxyribose analogues of the complement to microRNA-143 (miRNA-143). The system detects single nucleotide mismatches with high specificity, and has a limit of detection of 1.5 femtomoles. The system is capable of detecting miRNA-143 in cancer cell lysates, allowing for the discrimination between the MCF-7 (less aggressive) and MDA-MB-231 (more aggressive) cell lines. We propose that DMF-ECL represents a valuable new tool in the microfluidics toolbox for a wide variety of applications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Dopamine (DA) is a classical neurotransmitter and dysfunction in its synaptic handling underlies many neurological disorders, including addiction, depression, and neurodegeneration. A key to understanding DA dysfunction is the accurate measurement of dopamine uptake by dopaminergic neurons. Current methods that allow for the analysis of dopamine uptake rely on standard multiwell-plate based ELISA, or on carbon-fibre microelectrodes used in in vivo recording techniques. The former suffers from challenges associated with automation and analyte degradation, while the latter has low throughput and is not ideal for laboratory screening. In response to these challenges, we introduce a digital microfluidic platform to evaluate dopamine homeostasis in in vitro neuron culture. The method features voltammetric dopamine sensors with limit of detection of 30 nM integrated with cell culture sites for multi-day neuron culture and differentiation. We demonstrate the utility of the new technique for DA uptake assays featuring in-line culture and analysis, with a determination of uptake of approximately ∼32 fmol in 10 min per virtual microwell (each containing ∼200 differentiated SH-SY5Y cells). We propose that future generations of this technique will be useful for drug discovery for neurodegenerative disease as well as for a wide range of applications that would benefit from integrated cell culture and electroanalysis.

Electrochemistry, biosensors and microfluidics are popular research topics that have attracted widespread attention from chemists, biologists, physicists, and engineers. Here, we introduce the basic concepts and recent histories of electrochemistry, biosensors, and microfluidics, and describe how they are combining to form new application-areas, including so-called "point-of-care" systems in which measurements traditionally performed in a laboratory are moved into the field. We propose that this review can serve both as a useful starting-point for researchers who are new to these topics, as well as being a compendium of the current state-of-the art for experts in these evolving areas.

An initial investigation of the fabrication of a novel biosensor utilizing toll-like receptor 5 (TLR5) has been conducted. The detection assay using this sensor platform has been carried out using two complementary electrochemical techniques. The electrochemical properties of the modified bare gold surface following TLR5 immobilization were characterized. The electrochemical response to changes in the sensor film resistance and electron charge-transfer permittivity triggered by independent exposures to flagellins from Salmonella typhimurium (S. typhimurium) and Bacillus subtilis (B. subtilis) were examined and observed. The quantified film resistance data gathered using electrochemical impedance spectroscopy (EIS) over a macroscopic scale are in significant agreement with the corresponding electron charge-transfer permittivity measured locally by scanning electrochemical microscopy (SECM). Unlike other sensors that exploit pathogen recognition elements, TLR5 biosensors have the potential to carry out broad-spectrum detection of flagellated bacterial pathogens in near real time. This broad-spectrum detection platform is a significant step toward the development of fast, inexpensive clinical tools for early warning diagnoses and immediate on-site treatment.

Digital microfluidics (DMF) has emerged as a popular format for implementing quantitative immunoassays for diagnostic biomarkers. All previous reports of such assays have relied on optical detection; here, we introduce the first digital microfluidic immunoassay relying on electrochemical detection. In this system, an indium tin oxide (ITO) based DMF top plate was modified to include gold sensing electrodes and silver counter/pseudoreference electrodes suitable for in-line amperometric measurements. A thyroid stimulating hormone (TSH) immunoassay procedure was developed relying on magnetic microparticles conjugated with primary antibody (Ab1). Antigen molecules are captured followed by capture of a secondary antibody (Ab2) conjugated with horseradish peroxidase enzyme (HRP). HRP catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) which can be detected amperometrically. The limit of detection of the technique (2.4 μIU mL(-1)) is compatible with clinical applications; moreover, the simplicity and the small size of the detector suggest utility in the future for portable analysis.

Digital microfluidics (DMF) is an emerging technique for manipulating small volumes of liquids. DMF is particularly well suited for analytical applications as it allows automated handling of discrete samples, and it has been integrated with several inline analysis techniques. However, examples of the integration of DMF with electroanalytical methods are notably scarce, and those that have been reported rely on external electrodes that impose limitations on complexity. To combine the full capabilities of DMF with voltammetry, we designed a platform featuring a three-electrode electrochemical cell integrated in a microfabricated DMF device, removing the need for external electrodes and allowing for complete droplet control. The performance of the DMF/voltammetry system is comparable to that of a commercial screen printed electrode, and the new platform was applied to generating a calibration series for acetaminophen with a limit of detection of 76 μM and good precision (4% average RSD), all with minimal human intervention. We propose that this platform and variations thereof may be a useful new tool for microscale electroanalysis and will be a complementary system to existing inline analysis techniques for DMF.

High throughput DNA basepair mismatch detection is an ultimate goal for earlier and point-of-care diagnostics. However, the size of a target sequence on single nucleotide mismatch detection will critically impact the design of sensors in future. To study the potential impact of target size, the probe and target strands of unequal size were hybridized in the absence and presence of single nucleotide mismatches along the sequence. After hybridization, the shorter target sequences form overhangs in the probe strand while longer target sequences form overhangs in the complementary strand. The resulting double stranded DNA hybrids were printed on gold surfaces and the electrochemical response of the films was studied by scanning electrochemical microscopy without signal amplification and label. The redox mediator, [Fe(CN)(6)](4-), experiences lower repulsion in the vicinity of mismatch containing ds-DNA films, which ultimately manifests into higher feedback current regardless of the size and hybridization position of the complementary strands. Kinetic rate constants monitored right above the ds-DNA films show k(0) = 4.5 ± 0.1 × 10(-5) cm s(-1) for the short sequence hybridized at the upper portion of the probe while k(0) = 4.1 ± 0.2 × 10(-5) cm s(-1) for longer complementary strands which has only top overhang. It suggests that hybridization position is important for mismatch detection in short complementary stands. However, in longer complementary strands, mismatches are easily detectable in the absence of bottom overhangs.

While there are a number of electrochemical methods reported that enable the detection of single nucleotide mismatches, the determination of mismatch position in a double stranded DNA remains an unsolved challenge. Using a model system, we systematically explored the electrochemical response of all possible positions of single nucleotide mismatches in a set of 25-mer DNA films. These ds-DNA sequences each with a single mismatch at one of the twenty-five positions were bound to gold surfaces through a Au-S linkage and analyzed by electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) in the absence and presence of Zn(2+). We expected a unique response from each mismatched sequence in order to discriminate the mismatch positions. A pattern emerges between the electrochemical signals and mismatch positions. The positions can be grouped broadly into positions that exhibit large differences between matched and mismatched DNA (around positions 5 and 9) and those that exhibit smaller differences (around positions 1, 13 and 23) in the charge transfer resistance ΔR(ct), evaluated by EIS, and the apparent rate constant k(0), evaluated by SECM. To the best of our knowledge, this is the first study evaluating the electrochemical response of a single nucleotide mismatch as a function of mismatch positions along an oligonucleotide sequence.

Our studies show that electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) of films of ds-DNA on gold allow us to distinguish between mitochondrial DNA fragments of the cytochrome c(1) oxidase (mt-Cox1) of three related species of the subfamily 'Bovinae' (Bos taurus, Bison bison, and Bison bonasus). In EIS, a perfectly matched DNA gives rise to a considerably larger charge transfer resistance R(ct) compared to mismatched pairings. Differences in charge transfer resistance, ΔR(ct), before and after the addition of Zn(2+) ions provide an additional tool for identification. In addition, all ds-DNA films were studied by SECM and their kinetic parameters were determined. Perfectly matched ds-DNAs are readily distinguished from mismatched duplexes by their lower rate constants. Our system can be used multiple times by dehybridization and rehybridization of capture strands up to the 250 pmole level.

While oligonucleotide hybridization and effects of nucleobase mismatches have been the intense focus of a number of electrochemical studies, the effects of the target strand length on the electrochemical response of oligonucleotide films have not been addressed yet. In this report, we have studied the electrochemical impedance of the oligonucleotide films having overhangs on either the target or the surface bound capture strand. Each system gives different impedance responses, which were interpreted with the help of modified Randles' equivalent. Results indicate that comparable sizes of target and capture strands ensure the higher hybridization efficiency and film order. The presence of nucleobase overhangs at the bottom of the film causes lower changes in charge transfer resistance (ΔR(CT)) after hybridization due to lower hybridization efficiency and presumably non-uniformity in the film. Nucleobase overhangs at the top of the film result in higher ΔR(CT) due to higher film order and accumulation of negative charges but appear not to cause any steric congestion.

Electrochemical impedance spectroscopy (EIS) has been used as an ultrasensitive tool for label-free detection of single-nucleotide mismatches in double-stranded DNA (ds-DNA) films. In this study, we have explored the effects of the position and of the type of single-nucleotide mismatch in ds-DNA on gold surfaces and were able to distinguish mismatch positions and mismatch pairs. The single-nucleotide mismatches A-C, A-A and A-G were introduced at three positions within the sequence in bottom, middle and top positions of ds-DNA, the films were studied by EIS, and the impedance results were interpreted with the help of equivalent circuits. The DeltaR(ct), the difference in charge transfer resistance before and after the addition of Zn(2+), was used to distinguish single-nucleotide mismatch within the DNA sequences. Importantly, the mismatch pair is easily distinguishable at the middle position. A purine-pyrimidine mismatch can be distinguished from purine-purine mismatch by its lower DeltaR(ct) value. In addition, all ds-DNA films were studied by scanning electrochemical microscopy in the absence and presence of Zn(2+), allowing us to distinguish a range of mismatched films from matched ds-DNA film.

DNA-wrapped halloysite nanotubes were obtained by a mechanochemical reaction in the solid state. The characterization by scanning electron microscopy showed that the nanotubes were cut into shorter lengths and were completely covered with DNA. This resulted in a high aqueous solubility of the product with stability of the solution for about 6 weeks. The nanotubes were cut to different fractions with lengths of 200-400 nm (30-40%), 400-600 nm (10-20%) and 600-800 nm (5-10%) after ball milling. FTIR spectroscopic analysis shows that the DNA in the product remained intact. This straightforward technique for obtaining water-soluble halloysite nanotubes by a solid-state reaction has great potential for biomedical applications of nanotubes.