ABSTRACT
Antibacterial formulations based on zinc oxide nanoparticles (ZnO NPs) are widely used for antibiotic replacement in veterinary medicine and animal nutrition. However, the undesired environmental impact of ZnO NPs triggers a search for alternative, environmentally safer solutions. Here, we show that Zn2+ in its ionic form is a more eco-friendly antibacterial, and its biocidal action rivals that of ZnO NPs (<100 nm size), with a minimal biocidal concentration being 41(82) µg mL-1 vs 5 µg mL-1 of ZnO NPs, as determined for 103(106) CFU mL-1 E. coli. We demonstrate that the biocidal activity of Zn2+ ions is primarily associated with their uptake by E. coli and spontaneous in vivo transformation into insoluble ZnO nanocomposites at an internal bacterial pH of 7.7. Formed in vivo nanocomposite then damages E. coli membrane and intracellular components from the inside, by forming insoluble biocomposites, whose formation can also trigger ZnO characteristic reactions damaging the cells (e.g., by generation of high-potential reactive oxygen species). Our study defines a special route in which Zn2+ metal ions induce the death of bacterial cells, which might be common to other metal ions capable of forming semiconductor oxides and insoluble hydroxides at a slightly alkaline intracellular pH of some bacteria.
Subject(s)
Anti-Bacterial Agents , Escherichia coli , Zinc Oxide , Zinc Oxide/chemistry , Zinc Oxide/pharmacology , Escherichia coli/drug effects , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Metal Nanoparticles/chemistry , Metal Nanoparticles/toxicity , Zinc/chemistry , Zinc/pharmacology , Ions/chemistry , Microbial Sensitivity Tests , Reactive Oxygen Species/metabolism , Hydrogen-Ion Concentration , Nanocomposites/chemistryABSTRACT
Pathogen-triggered infections are the most severe global threat to human health, and to provide their timely treatment and prevention, robust methods for rapid and reliable identification of pathogenic microorganisms are required. Here, we have developed a fast and inexpensive electrocatalytic aptamer assay enabling specific and ultrasensitive detection of E. coli. E. coli, a biomarker of environmental contamination and infections, was captured on the mixed aptamer/thiolated PEG self-assembled monolayers formed on electrochemically pre-treated gold screen-printed electrodes (SPE). Signals from aptamer - E. coli binding were amplified by electrocatalytic reduction of ferricyanide mediated by methylene blue (MB) adsorbed on bacterial and aptamer surfaces. PEG operated as an antifouling agent and inhibited direct (not MB-mediated) discharge of ferricyanide. The assay allowed from 10 to 1000 CFU mL-1E. coli detection in 30 min, with no interference from B. subtilis, in buffer and artificial urine samples. This electrocatalytic approach is fast, specific, sensitive, and can be used directly in in-field and point-of-care applications for analysis of bacteria in human environment.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Humans , Methylene Blue/chemistry , Escherichia coli , Biosensing Techniques/methods , Aptamers, Nucleotide/chemistry , Electrochemical Techniques/methods , Ferricyanides , Gold/chemistry , Electrodes , Limit of DetectionABSTRACT
Non-invasive liquid biopsy assays for blood-circulating biomarkers of cancer allow both its early diagnosis and treatment monitoring. Here, we assessed serum levels of protein HER-2/neu, overexpressed in a number of aggressive cancers, by the cellulase-linked sandwich bioassay on magnetic beads. Instead of traditional antibodies we used inexpensive reporter and capture aptamer sequences, transforming the enzyme-linked immuno-sorbent assay (ELISA) into an enzyme-linked aptamer-sorbent assay (ELASA). The reporter aptamer was conjugated to cellulase, whose digestion of nitrocellulose film electrodes resulted in the electrochemical signal change. ELASA, optimized relative aptamer lengths (dimer vs monomer and trimer), and assay steps allowed 0.1 fM detection of HER-2/neu in the 10% human serum in 1.3 h. Urokinase plasminogen activator and thrombin as well as human serum albumin did not interfere, and liquid biopsy analysis of serum HER-2/neu was similarly robust but 4 times faster and 300 times cheaper than both electrochemical and optical ELISA. Simplicity and low cost of cellulase-linked ELASA makes it a perspective diagnostic tool for fast and accurate liquid biopsy detection of HER-2/neu and of other proteins for which aptamers are available.
ABSTRACT
Prevention of food spoilage, environmental bio-contamination, and pathogenic infections requires rapid and sensitive bacterial detection systems. Among microbial communities, the bacterial strain of Escherichia coli is most widespread, with pathogenic and non-pathogenic strains being biomarkers of bacterial contamination. Here, we have developed a fM-sensitive, simple, and robust electrocatalytically-amplified assay facilitating specific detection of E.coli 23S ribosomal rRNA, in the total RNA sample, after its site-specific cleavage by RNase H enzyme. Gold screen-printed electrodes (SPE) were electrochemically pre-treated to be productively modified with a methylene-blue (MB) - labelled hairpin DNA probes, which hybridization with the E. coli-specific DNA placed MB in the top region of the DNA duplex. The formed duplex acted as an electrical wire, mediating electron transfer from the gold electrode to the DNA-intercalated MB, and further to ferricyanide in solution, enabling its electrocatalytic reduction otherwise impeded on the hairpin-modified SPEs. The assay facilitated 20 min 1 fM detection of both synthetic E. coli DNA and 23S rRNA isolated from E.coli (equivalent to 15 CFU mL-1), and can be extended to fM analysis of nucleic acids isolated from any other bacteria.
Subject(s)
Biosensing Techniques , RNA, Ribosomal, 23S , RNA, Ribosomal, 23S/genetics , Escherichia coli/genetics , DNA/chemistry , Gold/chemistryABSTRACT
The discovery of new molecular biomarkers of cancer during the last decades and the development of new diagnostic devices exploiting those have significantly contributed to the clinical analysis of cancer and to improve the outcomes. Among those, liquid biopsy sensors exploiting aptamers for the detection of cancer biomarkers in body fluids are useful and accurate tools for a fast and inexpensive non-invasive screening of population. The incorporation of aptamers in electrochemical sandwich biosensors using enzyme labels, a so-called ELASA, has demonstrated its utility to improve the detection schemes. In this review, we overview the existing ELASA assays for numerous cancer biomarkers as alternatives to the traditional ELISA and discuss their possibilities to reach the market, currently dominated by optical immunoassays.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Neoplasms , Aptamers, Nucleotide/chemistry , Enzyme-Linked Immunosorbent Assay , Immunoassay , Biomarkers, Tumor , Electrochemical Techniques , Neoplasms/diagnosisABSTRACT
Orientated coupling of redox enzymes to electrodes by their reconstitution onto redox cofactors, such as hemin conjugated to self-assembled monolayers (SAMs) formed on the electrodes, poses the requirements for a SAM design enabling reconstitution. We show that the kinetics of electron transfer (ET) in binary SAMs of alkanethiols on gold composed of in situ hemin-conjugated 11-amino-1-undecanethiol (AUT) and diluting OH-terminated alkanethiols with 11, 6, and 2 methylene groups (MC11OH, MC6OH, and MC2OH) depends on both the SAM composition and surface density of hemin, Γheme. In AUT/MC11OH SAMs composed of equal linker/diluent lengths, the heterogeneous ET rate constant ks decreased with the Γheme and varied between 70 and 500 s-1. For shorter diluents, the ks of 245-330 s-1 (C6) and 300-340 s-1 (C2) showed a little (if any) Γheme dependence. In AUT/MC11OH SAMs, the increasing Γheme resulted in the steric crowding of hemin species and their neighboring lateral interactions in the plane of hemin localization, affecting the potential distribution at the SAM/electrode interface and inducing local electrostatic effects interfering with hemin oxidation. In AUT/MC6OH and AUT/MC2OH SAMs, hemin discharged at the plane of the closest approach to the gold surface, equal to the diluent length and permeable to electrolyte ions, which lessened those effects. All studied binary SAMs provided steric hindrance for protein reconstitution on the hemin cofactor conjugated to the extended AUT linker. Further use of SAM-modified electrodes with the covalently attached hemin as interfaces for heme proteins' reconstitution should consider SAMs with loosely dispersed redox centers terminating more rigid molecular wires. Such wires place hemin at fixed distances from the electrode surface and thus ensure the interfacial properties required for the effective on-surface reconstitution of proteins and enzymes.
Subject(s)
Gold , Hemeproteins , Electrodes , Electrons , Heme , HeminABSTRACT
Liquid biopsy assays for tumour biomarkers circulating in blood are perspective non-invasive tools for cancer diagnosis and treatment monitoring. Here, we suggest a simple, 1 h long electrochemical DNAzyme-linked aptamer- and immuno-sandwich magnetic assay for analysis of serum HER-2/neu protein overexpressed in several aggressive cancers. In the assay, we used a covalent hemin-guanine quadruplex (G4) complex as a novel O2-dependent electrocatalytic label that allowed 10 fM (aptamer-aptamer) and 1 fM (aptamer-antibody) detection of HER-2/neu in human serum. The O2 reactivity of the aptamer-conjugated label was detected at high-surface-area graphite electrodes displaying a high efficiency of O2 reduction electro-catalyzed by this DNAzyme. In contrast to the recognised H2O2 reactivity, the O2 reactivity of the covalent hemin/G4 complex depended only on ambient O2 present in solutions, and did not require adding such traditional reagents as hemin and H2O2, and solution de-aeration. Human serum albumin, urokinase plasminogen activator and thrombin did not interfere, and the assay was used for analysis of basal serum levels of HER-2/neu. Due to the simplicity and low cost, sandwich assays exploiting O2-linked electrocatalysis by the covalent hemin-G4 complexes represent a more advanced electrochemical ELISA platform for ultrasensitive and fast detection of low concentrations of proteins in complex biological matrices.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , DNA, Catalytic , G-Quadruplexes , Aptamers, Nucleotide/chemistry , Biological Assay , DNA, Catalytic/chemistry , Hemin/chemistry , Humans , Hydrogen Peroxide/chemistry , Limit of Detection , Magnetic Phenomena , OxygenABSTRACT
Serine protease thrombin is a strong neurotoxin produced by the injured brain and an Alzheimer's disease biomarker. To address its point-of-care testing (POCT), we adapted the O2-dependent aptamer assay for thrombin to gold screen-printed electrodes (Au SPE). The assay exploits reagentless (with no mediators) electrocatalytic activity of hemin-G4 DNAzyme in O2 reduction. Au SPEs modified with thiolated hemin-conjugated aptamer and PEG showed enhanced electrocatalytic activity in O2 reduction upon thrombin binding to the aptamer, then folding into the electroactive hemin-G4 DNAzyme structure. 0.5 fM thrombin were detected in aerated PBS and artificial cerebrospinal fluid, correspondingly, with the logarithmic linear range extending to 100 fM; dopamine, and uric and ascorbic acids did not interfere with electroanalysis. The disposable aptasensor met basic POCT requirements, with a minimal shelf life of 3 days. However, the reactivity and suitability of the Au SPE surface for thiol binding and electrocatalysis required special surface pre-treatment and modification protocols, and the fundamental problem of a long-term stability of thiol modification on gold should be addressed for practical applications of Au SPE-based apatasensors in POCT.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , DNA, Catalytic , G-Quadruplexes , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , DNA, Catalytic/chemistry , Electrochemical Techniques/methods , Electrodes , Gold/chemistry , Hemin/chemistry , Limit of Detection , Sulfhydryl Compounds , Thrombin/analysisABSTRACT
Thrombin, a serine protease playing a central role in the coagulation cascade reactions and a potent neurotoxin produced by injured brain endothelial cells, is a recognized cardiac biomarker and a critical biomarker for Alzheimer's disease development. Both in vivo and in vitro, its low physiological concentrations and nonspecific binding of other components of physiological fluids complicate electroanalysis of thrombin. Here, femtomolar levels of thrombin in serum and an artificial cerebrospinal fluid (CSF) were detected by the indicator-free electrochemical methodology exploiting the O2 reduction reaction directly, with no electron transfer mediators, electrocatalyzed by the covalent G4-hemin DNAzyme complex naturally self-assembling upon thrombin binding to the hemin-modified 29-mer DNA aptamer sequence tethered to gold via an alkanethiol linker. Coadsorbed PEG inhibited nonspecific protein binding and allowed the sought signal resolution. The proposed assay exploiting the "oxidase" activity of G4-hemin DNAzyme does not require any coreactants necessary for the traditional peroxidase activity-based assays with this DNAzyme, such as H2O2 and redox mediators, or solution deaeration and allows fast, overall 30 min analysis of thrombin in aerated buffer, CSF, and 1% human serum solutions. This pioneer approach exploiting the oxidase activity G4-hemin DNAzyme is simple, sensitive, and selective and represents a new tool for ultrasensitive electrocatalytic assays based on simple and efficient O2-dependent DNAzyme labels.
Subject(s)
Biosensing Techniques/methods , Electrochemical Techniques/methods , Thrombin/analysis , Alzheimer Disease/diagnosis , Alzheimer Disease/metabolism , Biomarkers/analysis , DNA, Catalytic/chemistry , Hemin/chemistry , Humans , MaleABSTRACT
Improved outcomes for many types of cancer achieved during recent years is due, among other factors, to the earlier detection of tumours and the greater availability of screening tests. With this, non-invasive, fast and accurate diagnostic devices for cancer diagnosis strongly improve the quality of healthcare by delivering screening results in the most cost-effective and safe way. Biosensors for cancer diagnostics exploiting aptamers offer several important advantages over traditional antibodies-based assays, such as the in-vitro aptamer production, their inexpensive and easy chemical synthesis and modification, and excellent thermal stability. On the other hand, electrochemical biosensing approaches allow sensitive, accurate and inexpensive way of sensing, due to the rapid detection with lower costs, smaller equipment size and lower power requirements. This review presents an up-to-date assessment of the recent design strategies and analytical performance of the electrochemical aptamer-based biosensors for cancer diagnosis and their future perspectives in cancer diagnostics.
Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Neoplasms , Antibodies , Electrochemical Techniques , Humans , Neoplasms/diagnosisABSTRACT
Microbiological safety of the human environment and health needs advanced monitoring tools both for the specific detection of bacteria in complex biological matrices, often in the presence of excessive amounts of other bacterial species, and for bacteria quantification at a single cell level. Here, we discuss the existing electrochemical approaches for bacterial analysis that are based on the biospecific recognition of whole bacterial cells. Perspectives of such assays applications as emergency-use biosensors for quick analysis of trace levels of bacteria by minimally trained personnel are argued.
Subject(s)
Aptamers, Nucleotide , Bacteria , Biosensing Techniques , Electrochemical Techniques , HumansABSTRACT
Pathogen-associated infections represent one of the major threats to human health and require reliable methods for immediate and robust identification of pathogenic microorganisms. Here, an inexpensive cellulase-linked immunomagnetic methodology was developed for the specific and ultrasensitive analysis of bacteria at their single-cell levels within a 3 h procedure. Detection of a model bacterium, Escherichia coli, was performed in a sandwich reaction with E. coli-specific either aptamer or antibody (Ab)-modified magnetic beads (MBs) and Ab/aptamer reporter molecules linked to cellulase. The cellulase-labeled immuno-aptamer sandwich applied onto nitrocellulose-film-modified electrodes digested the film and changed its electrical conductivity. Electrode's chronocoulometric responses at 0.3 V, in the absence of any redox indicators, allowed a single E. coli cell detection and from 1 to 4 × 104 CFU mL-1 E. coli quantification. No interference/cross-reactivity from Salmonella enteritidis, Enterobacter agglomerans, Pseudomonas putida, Staphylococcus aureus, and Bacillus subtilis was observed when the assay was performed on Ab-modified MBs, and E. coli could be quantified in tap water and milk. This electrochemically label-free methodology is sufficiently fast, highly specific, and sensitive to be used in direct in-field applications. The assay can be adapted for specific detection of other bacterial strains of either the same or different species and offers new analytical tools for fast, specific, and reliable analysis of bacteria in the clinic, food, and environment.
Subject(s)
Cellulase/metabolism , Escherichia coli/isolation & purification , Immunomagnetic Separation , Bacillus subtilis/cytology , Bacillus subtilis/isolation & purification , Bacillus subtilis/metabolism , Cellulase/chemistry , Electrodes , Enterobacter/cytology , Enterobacter/isolation & purification , Enterobacter/metabolism , Escherichia coli/cytology , Escherichia coli/metabolism , Pseudomonas putida/cytology , Pseudomonas putida/isolation & purification , Pseudomonas putida/metabolism , Salmonella enteritidis/cytology , Salmonella enteritidis/isolation & purification , Salmonella enteritidis/metabolism , Single-Cell Analysis , Staphylococcus aureus/cytology , Staphylococcus aureus/isolation & purification , Staphylococcus aureus/metabolismABSTRACT
The ability of the DNA double helix to transport electrons underlies many life-centered biological processes and bio-electronic applications. However, there is little consensus on how efficiently the base pair π-stacks of DNA mediate electron transport. This minireview scrutinizes the current state-of-the-art knowledge on electron transfer (ET) properties of DNA and its long-range ability to transfer (mediate) electrical signals at electrified interfaces, without being oxidized or reduced. Complex changes an electric field induces in the DNA structure and its electronic properties govern the efficiency of DNA-mediated ET at electrodes and allow addressing the existing phenomenological riddles, while recently discovered rectifying properties of DNA contribute both to our understanding of DNA's ET in living systems and to advances in molecular bioelectronics.
Subject(s)
DNA/chemistry , Electric Conductivity , Electron Transport , Molecular StructureABSTRACT
In cancer diagnostics, specific analysis of blood-circulating proteins biomarkers of cancer is often complicated both by their inherently low concentrations and by strong interference from serum/blood proteins. Here, we report a simple and robust electrochemical cellulase-linked sandwich assay on magnetic beads (MBs) for fM-sensitive analysis of the Human Epidermal growth factor Receptor-2 HER-2/neu protein that is over-expressed in most aggressive breast cancers. In the assay, a sandwich is assembled by capturing HER-2/neu on either antibody (Ab) or aptamer-modified MBs accompanied by reaction with the second Ab or aptamer labelled with cellulase. On application of the sandwiches assembled on MBs onto a cost-effective graphite electrode modified with an insulating nitrocellulose film, the cellulase label digests the film. This results in the pronounced changes in the electrical properties of the modified electrodes. The chronocoulometrically-measured extent of the produced changes was proportional to the 10-15-10-10â¯M HER-2/neu in the analyzed samples, and down to 1â¯fM of HER-2/neu could be detected in human serum samples in an overall less than 3â¯h assay. The developed simple and electrochemically label-free methodology is general and can be easily adapted for testing of any other protein.
Subject(s)
Biomarkers, Tumor/blood , Electrochemical Techniques/methods , Enzyme-Linked Immunosorbent Assay/methods , Receptor, ErbB-2/blood , Aptamers, Nucleotide/chemistry , Base Sequence , Cellulase/chemistry , DNA/chemistry , Electrochemical Techniques/instrumentation , Electrodes , Graphite/chemistry , Humans , Magnetic PhenomenaABSTRACT
Specific analysis of such neurotransmitters as dopamine by the aptamer electrodes in biological fluids is detrimentally affected by nonspecific adsorption of media, particularly pronounced at positive charges of the electrode surface at which dopamine oxidizes. Here, we show that dopamine analysis at the RNA-aptamer/cysteamine-modified electrodes is strongly inhibited in undiluted human serum and blood due to nonspecific interfacial adsorption of serum and blood components. We demonstrate that nonspecific adsorption of serum proteins (but not of blood components) could be minimized when analysis is performed in a flow and injections of serum samples are followed by washing steps in a phosphate buffer solution (PBS) carrier. Under those conditions, the dopamine-aptamer binding affinity in whole human serum of (1.9 ± 0.3) × 104 M-1 s-1 was comparable to the (3.7 ± 0.3) × 104 M-1 s-1 found in PBS, and the dopamine oxidation signal linearly depended on the dopamine concentration, providing a sensitivity of analysis of 73 ± 3 nA µM-1 cm-2 and a LOD of 114 ± 8 nM. The flow-injection apatmer-electrode system was used for direct analysis of basal levels of dopamine in undiluted human serum samples, without using any physical separators (membranes) or filtration procedures. The results suggest a simple strategy for combatting biosurface fouling, otherwise most pronounced at positive electrode potentials used for dopamine detection, and assist in designing more efficient antifouling strategies for biomedical applications.
Subject(s)
Adsorption/physiology , Aptamers, Nucleotide/blood , Dopamine/blood , Electrodes , Biosensing Techniques/methods , Electrochemical Techniques/methods , Humans , Metal Nanoparticles/analysis , Oxidation-ReductionABSTRACT
Electrical properties of self-assembling DNA nanostructures underlie the paradigm of nanoscale bioelectronics, and as such require clear understanding. DNA-mediated electron transfer (ET) from a gold electrode to DNA-bound Methylene Blue (MB) shows directional preference, and it is sequence-specific. During the electrocatalytic reduction of [Fe(CN)6 ]3- catalyzed by DNA-bound MB, the ET rate constant for DNA-mediated reduction of MB reaches (1.32±0.2)103 and (7.09±0.4)103 â s-1 for (dGdC)20 and (dAdT)25 duplexes. The backward oxidation process is less efficient, making the DNA duplex a molecular rectifier. Lower rates of ET via (dGdC)20 agree well with its disturbed π-stacked sub-molecular structure. Such direction- and sequence-specific ET may be implicated in DNA oxidative damage and repair, and be relevant to other polarized surfaces, such as cell membranes and biomolecular interfaces.
ABSTRACT
Ability of the DNA double helix to transport electrons is its critical feature, underlying a number of important biological and biotechnological processes. Here, we show that electron transfer (ET) from the gold electrode to the DNA-bound methylene blue (MB) mediated by the DNA base-pair π-stack is less efficient in (dGdC)-rich duplexes compared to pure (dAdT) DNA. The ET rate constant ks extrapolated to the DNA surface coverage ΓDNA â 0 is 121 ± 8 s-1 for (dAdT)25, being almost twofold higher than 67 ± 3 s-1 shown for (dGdC)20, consistent with the electric-field-disturbed submolecular structure of the (dGdC)20 duplex earlier shown at electrified interfaces. DNA-mediated ET occurs both to MB intercalated and thus perfectly π-stacked into the (dGdC)20 duplex and to MB solely groove-bound to (dAdT)25. For both (dGdC)20 and (dAdT)25, ET is less efficient than ET in DNA duplexes of a mixed dA, dT, dG, dC composition. The results suggest new interpretations of the biological ET processes that may occur in dsDNA of different compositions at polarized interfaces.
Subject(s)
DNA/chemistry , Electrons , Gold/chemistry , Intercalating Agents/chemistry , Methylene Blue/chemistry , Electrochemical Techniques/instrumentation , Electrochemical Techniques/methods , Electrodes , Hexanols/chemistry , Kinetics , Osmolar Concentration , Sulfhydryl Compounds/chemistryABSTRACT
Electrochemical enzyme-linked sandwich assays on magnetic beads (MBs) refer to one of the most sensitive approaches for analysis of nonamplified nucleic acid samples, with redox enzymes being routinely used as labels. Here, we report a sensitive and inexpensive electrochemical nucleic acid sandwich assay on MBs that exploits a hydrolytic enzyme cellulase as a label, while MBs are used for preconcentration and bioseparation of analyzed samples. Binding of target DNA or RNA to capture DNA-modified MB triggers sandwich assembly and its labeling with cellulase. Application of the assembled sandwich to the electrodes covered with insulating nitrocellulose films induces film digestion by the cellulase label and pronounced changes in the electrical properties of the electrodes, the extent of the changes being proportional to the concentration of the analyzed nucleic acids. Down to 1 amol of Lactobacillus brevis specific synthetic DNA and rRNA isolated from L. brevis cells could be detected in 1 mL samples in the overall from 2 to 3 h assay. The assay is universal and can be adapted for point-of-care-testing and for in-field environmental and microbiomic analysis of unamplified samples of any DNA/RNA sequences.
Subject(s)
Cellulase/metabolism , DNA, Bacterial/analysis , Electrochemical Techniques/methods , RNA, Ribosomal/analysis , Cellulase/chemistry , Electrodes , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Ferricyanides/chemistry , Hydrolysis , Levilactobacillus brevis/genetics , Magnetics , Point-of-Care SystemsABSTRACT
A robust and sensitive electrochemical assay for chrononocoulometric detection of nucleic acids at a single nucleotide polymorphism (SNP) level has been developed. The assay exploits hybridization-induced conformational switching of gold-tethered TP53-specific 33-mer and truncated 20-mer hairpin DNA probes and methylene blue (MB) as an intercalating redox indicator. We show that by fine tuning of MB-DNA intercations the enhanced binding of MB to hybrids formed with a cancer-biomarker sequence can be achieved, and that results in robust "off-on" sensing of hybridization, while the stem-loop probe design allows minimized, independent of the DNA length background signals. Both DNA probes were sensitive to the presence of SNP in the targeted DNA sequence already at 10â¯pM. DNA levels, and the robust "off-on" discrimination of 10â¯pM perfectly-matched DNA from 50â¯nM SNP-containing DNA was achieved by time-adjusted chronocoulometry. This label-free hairpin DNA strategy allows systematic design of DNA assays for fast, robust and inexpensive genetic analysis in excessive mixtures of structurally-related DNA sequences and was used for specific analysis of prostate-cancer-realted cellular microRNA in total RNA samples isolated from LNCaP and BPH1 cells.
Subject(s)
Biosensing Techniques/methods , DNA Probes/chemistry , DNA/analysis , Electrochemical Techniques , Nucleic Acids/analysis , Polymorphism, Single Nucleotide , DNA/chemistry , DNA Probes/standards , Nucleic Acid Hybridization , Oxidation-ReductionABSTRACT
Electrical properties of DNA critically depend on the way DNA molecules are integrated within the electronics, particularly on DNA-electrode immobilization strategies. Here, we show that the rate of electron transport in DNA duplexes spacer-free tethered to gold via the adenosine terminal region (a dA10 tag) is enhanced compared to the hitherto reported DNA-metal electrode tethering chemistries. The rate of DNA-mediated electron transfer (ET) between the electrode and methylene blue intercalated into the dA10-tagged DNA duplex approached 361 s-1 at a ca. half-monolayer DNA surface coverage ΓDNA (with a linear regression limit of 670 s-1 at ΓDNA â 0), being 2.7-fold enhanced compared to phosphorothioated dA5* tethering (6-fold for the C6-alkanethiol linker representing an additional ET barrier). X-ray photoelectron spectroscopy evidenced dA10 binding to the Au surface via the purine N, whereas dA5* predominantly coordinated to the surface via sulfur atoms of phosphothioates. The latter apparently induces the DNA strand twist in the point of surface attachment affecting the local DNA conformation and, as a result, decreasing the ET rates through the duplex. Thus, a spacer-free DNA coupling to electrodes via dA10 tags thus allows a perspective design of DNA electronic circuits and sensors with advanced electronic properties and no implication from more expensive, synthetic linkers.