DNA Detection Method Based on the Two-Dimensional Aggregation and Selective Desorption of Nanoparticle Probes

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two-five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor's detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Label-free detection of DNA hybridization based on a novel functionalized conducting polymer

Using molecular light switch Ru complex Ru(bpy)(2)(dppz)(2+) and CdTe quantum dots (QDs), we have designed a label-free DNA fluorescent sensor for the detection of Ag(+) in aqueous solution.

The ability to prepare single-stranded chromosomal target DNA allows innovative uses of FISH technology for studies of chromosome organization. Standard FISH methodologies require functionally single-stranded DNAs in order to facilitate hybridization between the probe and the complementary chromosomal target sequence. This usually involves denaturation of double-stranded probes to induce temporary separation of the DNA strands. Strand-specific FISH (CO-FISH; Chromosome Orientation-FISH) involves selective removal of newly replicated strands from DNA of metaphase chromosomes which results in single-stranded target DNA. When single-stranded probes are then hybridized to such targets, the resulting strand-specific hybridization is capable of revealing a level of information previously unattainable at the cytogenetic level. Mammalian telomeric DNA consists of tandem repeats of the (TTAGGG) sequence, oriented 5′→3′ towards the termini of all vertebrate chromosomes. Based on this conserved structural organization, CO-FISH with a telomere probe reveals the absolute 5′→3′ orientation of DNA sequences with respect to the pter→qter direction of chromosomes. Development and various applications of CO-FISH will be discussed: detection of cryptic inversions, discrimination between telomeres produced by leading- versus lagging-strand synthesis, and replication timing of mammalian telomeres.

Oligonucleotide hybridization probes that fluoresce upon binding to complementary nucleic acid targets allow the real-time detection of DNA or RNA in homogeneous solution. The most commonly used probes rely on the distance-dependent interaction between a fluorophore and another label. Such dual-labeled oligonucleotides signal the change of the global conformation that accompanies duplex formation. However, undesired nonspecific binding events and/or probe degradation also lead to changes in the label-label distance and, thus, to ambiguities in fluorescence signaling. Herein, we introduce singly labeled DNA probes, "DNA FIT probes", that are designed to avoid false-positive signals. A thiazole orange (TO) intercalator dye serves as an artificial base in the DNA probe. The probes show little background because the attachment mode hinders 1) interactions of the "TO base" in cis with the disordered nucleobases of the single strand, and 2) intercalation of the "TO nucleotide" with double strands in trans. However, formation of the probe-target duplex enforces stacking and increases the fluorescence of the TO base. We explored open-chain and carbocyclic nucleotides. We show that the incorporation of the TO nucleotides has no effect on the thermal stability of the probe-target complexes. DNA and RNA targets provided up to 12-fold enhancements of the TO emission upon hybridization of DNA FIT probes. Experiments in cell media demonstrated that false-positive signaling was prevented when DNA FIT probes were used. Of note, DNA FIT probes tolerate a wide range of hybridization temperature; this enabled their application in quantitative polymerase chain reactions.

Most challenging in the development of DNA sensors is the ability to distinguish between fully complementary target ssDNA (single-strand DNA) and 1-mismatch ssDNA. To deal with this problem, we performed impedance spectroscopy on DNA-functionalized nanocrystalline diamond (NCD) layers during hybridization and denaturation. In both reactions, a difference in behavior was observed for 1-mismatch target DNA and complementary target DNA in real-time. During real-time hybridization, a decrease of the impedance was observed at lower frequencies when the complementary target DNA was added, while the addition of 1-mismatch target ssDNA caused no significant change. Fitting these results to an electrical circuit demonstrates that this is correlated with a decrease of the depletion zone in the space charge region of the diamond. During real-time denaturation, differentiation between 1-mismatch and complementary target DNA was possible at higher frequencies. Denaturation of complementary DNA showed the longest exponential decay time of the impedance, while the decay time during 1-mismatch denaturation was the shortest. The real-time hybridization and denaturation experiments were carried out on different NCD samples in various buffer solutions at temperatures between 20 and 80 degrees C. It was revealed that the best results were obtained using a Microhyb hybridization buffer at 80 degrees C and 10x PCR buffer at 30 degrees C for hybridization and 0.1 M NaOH at temperatures above 40 degrees C for denaturation. We demonstrate that the combination of real-time hybridization spectra and real-time denaturation spectra yield important information on the type of target. This approach may allow a reliable identification of the mismatch sequence, which is the most biologically relevant.

Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe

Lateral flow DNA biosensor for visual detection of nucleic acid with triple-helix DNA functionalized carbon nanotube

A morphologically complex cellulosic substance (e.g., commercial filter paper) was employed as a substrate for DNA immobilization and successive recognition. A uniform ultrathin zirconia gel film was first deposited on each cellulose nanofiber in bulk filter paper by a facile sol-gel process. Relying on the large surface area of filter paper and the strong affinity of zirconia for the phosphate group, terminal-phosphate probe DNA was abundantly immobilized on the zirconia-modified filter paper so as to convert the composite to a biofunctional material for the sensitive and repetitive recognition of the corresponding complementary target DNA on the nanomolar level. By contrast, in spite of the viability of the immobilization of the probe DNA and the recognition of target DNA on the quartz plate, the amount of captured probe DNA or recognized target DNA on such a flat substrate was much less than that captured or recognized on filter paper, resulting in a relatively insensitive recognition event. Moreover, control experiments on bare filter paper (without a zirconia nanocoating) suggested that the zirconia gel film was essential to probe DNA immobilization and subsequent target DNA recognition.

Cell-generated forces are a key player in cell biology, especially during cellular shape formation, migration, cancer development, and immune response. A new type of label-free smartphone-based electrochemical DNA sensor is developed here for cellular force measurement. When cells apply tension forces to the DNA sensors, the rapid rupture of DNA duplexes allows multiple redox reporters to reach the electrode and generate highly sensitive electrochemical signals. The sensitivity of these portable sensors can be further enhanced by incorporating a CRISPR-Cas12a system. Meanwhile, the threshold force values of these DNA-based sensors can be rationally tuned based on the force application geometries and also DNA intercalating agents. Overall, these highly sensitive, portable, cost-efficient, and easy-to-use electrochemical sensors can be powerful tools for detecting different cell-generated molecular forces.

A one-step electrochemical method for DNA detection that utilizes a peroxidase-mimicking DNAzyme amplified through PCR of target DNA

Detection of dengue target by generation of silver nanoclusters using cocktail probes and single probe assay.

An electrochemical nucleic acid biosensor typically involves the immobilization of DNA strands onto a surface, which serves as a recognition element for specific DNA or RNA target molecules.1-3 DNA strands can be single-stranded or double-stranded, and their immobilization onto a surface is crucial for sensing applications. This allows the formation of monolayers which is often used to prevent nonspecific adsorption of interfering molecules onto the surface. This layer acts as a barrier, allowing only specific interactions with the DNA target molecules or analytes of interest. Traditionally, redox mediators such as ferrocyanide/ ferricyanide that undergo reversible oxidation-reduction reactions are often used to detect hybridization of DNA targets with immobilized complementary DNA probes. Under these conditions, the heterogeneous charge transfer rate of redox mediators at the electrode surface is often a key factor taken into account to interpret the sensitivity and efficiency of the biosensor.3-5 In this work, with a set of experiments carried out by hydrodynamic voltammetry and impedance spectroscopy at channel microelectrodes, we propose a new approach supported by numerical modeling to highlight the influence of the mass transport of redox mediators on the response of DNA biosensors. Contrary to what is commonly accepted in the literature, we showed that the presence of DNA monolayers acting as blocking monolayers does not affect the charge transfer rate of redox mediators, regardless of the electrode accessibility (surface coverage). We demonstrated that under flow conditions, the current response of the biosensors was controlled by the mass transport rather than the charge transfer rate of redox mediators. This phenomenon was attributed to the nonlinear effects of diffusion-convection of the redox mediators at the remaining active sites, thereby affecting the overall kinetics of the transduction signal. These experiments, coupled with numerical calculations, allowed us to compare the performance of DNA biosensors obtained with different blocking monolayers and to evaluate the average size of the active sites. In this model, they were assumed to be small compared to the electrode size.This study constitutes an important advance in the field of sensors or biosensors, in the broad sense, as it points out the influence of parameters controlling mass transport and therefore the performance of electrochemical DNA biosensors, such as the density of DNA monolayers, the composition of the blocking agents, and size of holes (active sites). REFERENCES 1 Slinker, J. D.; Muren, N. B.; Gorodetsky, A. A.; Barton, J. K. Multiplexed DNA-Modified Electrodes. J Am Chem Soc 2010, 132 (8). 2 Horny, M. C.; Lazerges, M.; Siaugue, J. M.; Pallandre, A.; Rose, D.; Bedioui, F.; Deslouis, C.; Haghiri-Gosnet, A. M.; Gamby, J. Electrochemical DNA Biosensors Based on Long-Range Electron Transfer: Investigating the Efficiency of a Fluidic Channel Microelectrode Compared to an Ultramicroelectrode in a Two-Electrode Setup. Lab Chip 2016, 16 (22). 3 Zambry, N. S.; Awang, M. S.; Beh, K. K.; Hamzah, H. H.; Bustami, Y.; Obande, G. A.; Khalid, M. F.; Ozsoz, M.; Manaf, A. A.; Aziah, I. A Label-Free Electrochemical DNA Biosensor Used a Printed Circuit Board Gold Electrode (PCBGE) to Detect SARS-CoV-2 without Amplification. Lab Chip 2023, 23 (6). 4 Tersch, C.; Lisdat, F. Label-Free Detection of Protein-DNA Interactions Using Electrochemical Impedance Spectroscopy. Electrochim Acta 2011, 56 (22). 5 Cardoso, A. R.; Moreira, F. T. C.; Fernandes, R.; Sales, M. G. F. Novel and Simple Electrochemical Biosensor Monitoring Attomolar Levels of MiRNA-155 in Breast Cancer. Biosens Bioelectron 2016, 80.

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia. It results from a deficiency in a specific cytochrome P450, P450c21 (P450XXIA). The gene encoding this protein (CYP21B) and a closely linked pseudogene (CYP21A) are located in the HLA complex on chromosome 6p. Many mutant alleles are associated with deletions of CYP21B; we report the cloning and characterization of a nondeleted mutant CYP21B gene. This mutant gene is expressed on transfection into mouse Y1 adrenal cells, producing mRNA levels similar to those seen after transfection of the normal CYP21B gene. In codon 172 of the mutant gene, the normal codon ATC, encoding isoleucine, has been changed to AAC, encoding asparagine. This mutation is normally present in the CYP21A pseudogene, so that it may have been transferred to the mutant CYP21B gene by gene conversion. Hybridization of oligonucleotide probes corresponding to this and two other mutations normally present in CYP21A demonstrated that 4 out of 20 patients carried the codon 172 mutation; in one of these patients, the mutation was present as part of a larger gene conversion involving at least exons 3-6. Gene conversion may be a frequent cause of 21-hydroxylase deficiency alleles due to the presence of six chi-like sequences (GCTGGGG) in the CYP21 genes and the close proximity of the CYP21A pseudogene, which has several potentially deleterious mutations.

5202231 Method of sequencing of genomes by hybridization of oligonucleotide probes: Radoje T Drmanac, Radomir B Crkvenjakov, Toronto