A toehold-aided DNA recycling amplification technology was developed based on the combination of toehold-aided DNA recycling and the hemin/G-quadruplex label. The dsDNA formed between aptamer and DNA1 was first immobilized on magnetic beads. On addition of target analyte (exemplified here for riboflavin), the aptamer-riboflavin complex is formed and DNA1 is released by the beads. After magnetic separation, the supernatant containing the released DNA1 is added to a solution containing the hairpin capture DNA on magnetic beads. DNA1 will hybridize with the hairpin capture DNA via toehold binding and branch migration. This process will open the hairpin structure, and an external toehold is formed in the newly formed dsDNA. On addition of reporter DNA containing the G-quadruplex, it will interact with the formed dsDNA via toehold binding and branch migration, resulting in the releasing of DNA1 and capturing of reporter DNA on the magnetic beads. The released DNA1 will bind to another hairpin capture DNA which can start another round of DNA1 recycling. Chemiluminescence (CL) is generated by the G-quadruplex-hemin-luminol CL reaction system. Under optimal conditions, the calibration plot is linear in the 0.1 to 700 nM riboflavin concentration range, with a 30 pM detection limit (at a signal-to-noise ratio of 3). The method was successfully applied to the quantitation of riboflavin in spiked urine samples.
A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3? edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable.
Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.
The authors describe an electrochemical DNA nanosensor based on the use of single gold nanowire electrodes (AuNWEs). The probe DNA is immobilized on the AuNWE via Au-S bonds that are formed between thiol-terminated DNA and the gold surface. Single AuNWEs were prepared by an improved laser-assisted pulling method and hydrofluoric acid etching. The nanoelectrodes were characterized by cyclic voltammetry and COMSOL simulation. Square wave voltammetry was used to monitor the DNA hybridization event between probe DNA and target DNA by using Methylene Blue (MB) as an intercalator of dsDNA. Under optimal conditions, the peak current for MB (best measured at a potential of ?0.2 V vs. Ag/AgCl) increases linearly with the logarithm of the analyte concentration in the 1.0 f. to 10 nM range, with a 0.48 fmM detection limit at an S/N ratio of 3. The assay is highly selective, reproducible and stable. Considering the small overall dimensions and high sensitivity, this nanoelectrode potentially can be applied to in-vivo sensing of DNA inside living cells
Graphical abstract Schematic presentation of an electrochemical DNA nanosensor using single gold nanowire electrodes and based on the interaction of thiol-terminated DNA and gold surface. It was used to detect complementary DNA with high selectivity and sensitivity.
A study is presented on the binding kinetics and mechanism of the adsorption of dsDNA on citrate-capped gold nanoparticles (AuNPs). Methods include fluorescence titration, isothermal calorimetry (ITC) titration, dynamic light scattering and gel electrophoresis. It is found that the fluorescence of probe DNA (labeled with Rhodamine Green and measured at excitation/emission peaks of 498/531 nm) is quenched by addition of AuNPs. The Stern-Volmer quenching constant (Ksv) is 1.67?×?10^9 L·mol?1 at 308 K and drops with increasing temperature. The quenching mechanism is mainly static. The results of both fluorescence titrations and ITC show negative values for ΔH and ΔS values. This shows ion-induced dipole-dipole interaction to be the main attractive forces between dsDNA and AuNPs, while electrostatic interactions result in repulsion. The repulsive forces lead to a lower affinity between dsDNA and AuNPs (compared to single-strand DNA). It is also found that dsDNA can prevent the aggregation of AuNPs which is accompanied by a color change from red into blue. The visual detection limit with bare eyes for dsDNA1 is 36 pM. Based on these findings, a colorimetric method was developed to detect the proto-oncogene of serine/threonine-protein kinase B-Raf V600E point mutation in HT29, Ec109, A549, Huh-7 and SW480 cell lines.
Graphical abstract Schematic of the salt-induced aggregation of uncapped gold nanoparticles (AuNPs) which leads to a color change from red to blue. If the AuNPs are coated with dsDNA, aggregation is suppressed.
The authors report on a simple strategy for sensitive determination of the activity of terminal deoxynucleotidyl transferase (TdT) using copper nanoclusters (CuNCs) as fluorescent probes. TdT-polymerized long chain AT-rich DNA serves as a template for the synthesis of the CuNCs, and TdT activity is detected fluorometrically at excitation/emission wavelengths of 340/570 nm. The protocol relies on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs. The calibration plot is linear in the 0.7 to 14 U L?1 activity range, with a 60 mU L?1 detection limit (at a signal-to-noise ratio of 3). The protocol was applied to determine TdT activity in acute lymphatic leukemia cells. This approach is selective, simple, convenient and cost-efficient because a complex DNA sequence is not required. In our perception, the method provides a viable new platform for monitoring the activity and inhibition of TdT.
Graphical abstract Based on the target-triggered formation of dsDNA polymers and in-situ formation of CuNCs with strong fluorescence, a turn-on fluorescence assay for TdT activity is presented.
The authors have investigated (a) the self-assembly of single-stranded DNA (ssDNA) on glass surfaces, and (b) the interaction of DNA with liquid crystals (LCs) on solid surfaces. The results suggest that ssDNA (compared to dsDNA) on the solid interface causes particularly different orientations in LCs. The LC molecules assume a uniform homeotropic orientation on the surface with a typical surface ssDNA coverage of ~2.4 × 1012 molecules per square cm. Once complementary DNA is hybridized on the surface, the homotropic orientation of the LCs becomes disrupted. This orientation transition can be visually observed by using a crossed polarizer. The findings were exploiting to design an assay for target DNA (= analyte DNA) that has an ~0.1 nM detection limit. The assay is highly selective and can easily differentiate target DNA from single-base mismatch and non-complementary DNA. In our perception, it represents a powerful, label-free and portable DNA detection scheme.
Graphical abstract Schematic illustration of the mechanism for orientation behavior of a liquid crystal film supported on different surfaces. The homeotropic orientation of LC molecules was induced by ssDNA with appropriate surface coverage and was disrupted by ssDNA with lower or higher surface coverage or P1/T1 complex. 5CB: 4-Cyano-4′-pentylbiphenyl. TEA: Triethoxysilylbutyraldehyde.
An electrochemical biosensor for determination of DNA is described that is based on the reaction of regulated DNA (reg-DNA) first with substrated DNA (subs-DNA) to form a reaction intermediate. The intermediate binds target DNA (T) by hybridization and initiates a branch migration leading to the production of complex of substrated DNA and target DNA (TC). Once TC is produced, it reacts with assisted DNA (ass-DNA) through a toehold exchange mechanism, yielding the product complex of substrated DNA and assisted DNA (CS). The target is then released back into the solution and and catalyzes the next cycle of toehold-exchange with the reaction intermediate of substrated DNA and regulated DNA (CPR). Unlike in a conventional DNA toehold that is hardwired with the branch migration domain, the allosteric DNA toehold is designed into a reg-DNA which is independent of the branch migration domain. Under the optimal experimental conditions and at a working potential as low as 0.18 V, response to DNA is linear in the 1 fM to 1000 pM concentration range, and the detection limit is 0.83 fM. The assay is highly specific and can discriminate target DNA even from a single-base mismatch. It was applied to the analysis of DNA spiked plasma samples.
Graphical abstract Schematic illustration of the electrochemical strategy for target DNA detection based on regulation of DNA strand displacement using an allosteric DNA toehold strategy. It can be used to analyze DNA-spiked plasma samples and has a low detection limit of 0.83 fM.
A DNAzyme-embedded hyperbranched DNA dendrimer is used as a colorimetric signal amplifier in an ultrasensitive detection scheme for nucleic acids. The hyperbranched DNA dendrimers were constructed by single-step autonomous self-assembly of three structure-free DNA monomers. A cascade of self-assembly reactions between the first and second strands leads to the formation of linear DNA concatemers containing overhang flank fragments. The third strand, which bears a peroxidase-mimicking DNAzyme domain, serves as a bridge to trigger self-assembly between the first and second strands across the side chain direction. This results in a chain branching growth of the DNAzyme-embedded DNA dendrimer. This signal amplifier was incorporated into the streptavidin-biotin detection system which comprises an adaptor oligonucleotide and a biotinylated capture probe. The resulting platform is capable of detecting a nucleic acid target with an LOD as low as 0.8 fM. Such sensitivity is comparable if not superior to most of the reported enzyme-free (and even enzyme-assisted) signal amplification strategies. The DNA dendrimer based method is expected to provide a universal platform for extraordinary signal enhancement in detecting other nucleic acid biomarkers by altering the respective sequences of adaptor and capture probe.
Graphical abstract Schematic of an assembly of a DNAzyme-embedded hyperbranched DNA dendrimer which operates as a signal amplifier for nucleic acids detection. The nanostructure is constructed by autonomous self-assembly of three DNA monomers. Colored letters represent each domain, and complementary domains are marked by asterisk. Domain d represents the DNAzyme sequence.
The authors describe an electrochemical strategy for highly sensitive determination of ATP that involves (a) aptamer-based target recognition, (b) enzyme-free dendritic DNA nanoassembly amplification with multiplex binding of the biotin-strepavidin system, and (c) enzyme-amplified differential pulse voltammetric readout. In the presence of ATP, binding of ATP to the aptamer releases trigger DNA from the double-stranded complex between ATP aptamer and trigger DNA. The single-stranded thiolated capture probe, chemisorbed on the gold electrode surface, captures the released trigger DNA via hybridization. The toehold of the trigger DNA is recombined with one end of the first substrate DNA (1) which is on its other end biotinylated and blocked, with loops, by a counterstrand. The latter is removed by a complementary single-stranded helper (1) exposing two toeholds and two identical complimentary sequences for a second biotinylated substrate DNA (2). The latter, which is double-stranded except for the toehold, binds to one of these two sites. It is then stripped from its counter strand by another single-stranded helper DNA 2, exposing a toehold to bind another substrate DNA 1. On this substrate, another cycle with dentrimeric bransching can start.Substrate 1 with its two binding sites for substrate 2 initiates the assembly of dendritic DNA on the surface of the gold electrode, which finally possesses numerous biotins at the terminal ends of both of the associated substrate DNAs. Subsequent multiplex binding of streptavidinylated alkaline phosphatase and enzyme-amplified electrochemical readout leads to a highly sensitive electrochemical ATP aptasensor. If operated in the DPV mode, the current as measured at a typical working potential of 0.25 V (vs. Ag/AgCl) increases linearly over the 10 nM to 10 μM logarithmic ATP concentration range, and the detection limit is 5.8 nM (at an S/N ratio of 3). The assay is highly specific and reproducible. It was successfully applied to the detection of ATP in spiked human serum samples.
Graphical Abstract Schematic of the electrochemical strategy for adenosine triphosphate detection using aptamer-based target recognition and dendritic DNA nanoassembly amplification
We describe a colorimetric assay for mercury(II) ion. It is based on a hybridization chain reaction (HCR) and the use of Fe3O4@Au nanoparticles (NPs). Aptamers specific for Hg(II) were immobilized on the surface of the Fe3O4@AuNPs. The presence of Hg(II) inhibits the HCR process and this enables less Methylene Blue (MB) to intercalate into the dsDNA structure. After magnetic separation of the DNA-loaded NPs carrying Hg(II), the change in the absorbance of the residual MB solution is measured at 663 nm. The respective calibration plot is linear in the 1 to 300 nM concentration range, with a 0.7 nM detection limit (at a signal-to-noise ratio of 3). The method displays excellent selectivity over other metal ions. It was applied to the analysis of Hg(II) in spiked river water.
Graphical abstract Fe3O4@Au nanoparticles (NPs) were fabricated, then aptamers were modified on the surface of Fe3O4@AuNPs. The absence of Hg2+ leads to the formation of dsDNA polymers via hybridization chain reaction (HCR) process on the surface of Fe3O4@AuNPs, Methylene Blue (MB) intercalates into these DNA polymers, which can be easily separated from MB solution by applying a magnet, thereby inducing a color change of the MB solution.
The authors introduce a method for spatially arranged DNA immobilization on 10-nm gold nanoparticles (GNP) deposited on a silicon substrate carrying nanogapped interdigitated electrodes. The GNPs are covalently bound to the surface via silane chemistry, and the single steps of fabrication are monitored by FTIR spectroscopy and atomic force microscopy. This GNP deposition technique is shown to reduce the size of the nanogaps to 130 nm. FTIR also was used to monitor the immobilization of DNA on the surface of the interdigitated electrodes. This method allows DNA to be immobilized in a uniform and homogenous way. The utility of the method is demonstrated by immobilizing probe DNA on the surface and detecting target DNA specific for the human papilloma virus via fluorescence with a detection limit as low as 1 pM. In our perception, this method for GNP-mediated DNA immobilization enables high-performance sensing of a wide range of target (analyte) DNA.
Graphical abstract Schematic presentation of gold nanoparticle-mediated and spatially resolved deposition of DNA on nano-gapped interdigitated electrodes. The method was applied to the chemiluminescent determination of the human papillomavirus
The authors describe an SPR sensor chip coated with gold nanoparticles (AuNPs) that enables highly sensitive determination of genetically modified (GM) crops. Detection is based on localized surface plasmon resonance (LSPR) with its known sensitivity to even minute changes in refractive index. The device consists of a halogen light source, a light detector, and a cuvette cell that contains a sensor chip coated with AuNPs. It is operated in the transmission mode of the optical path to enhance the plasmonic signal. The sample solution containing target DNA (e.g. from the GM crop) is introduced into the cuvette with the sensor chip whose surface was functionalized with a capture DNA. Following a 30-min hybridization, the changes of the signal are recorded at 540 nm. The chip responds to target DNA in the 1 to 100 nM concentration range and has a 1 nM detection limit. Features of this sensor chip include a short reaction time, ease of handling, and portability, and this enables on-site detection and in-situ testing.
Graphical abstract A localized surface plasmon resonance (LSPR)-based nanoplasmonic spectroscopic device enabling a highly sensitive biosensor is developed for the detection of genetically modified (GM) DNA founded in Roundup Ready (RR) soybean.
Patients with prostate cancer and systemic lupus erythematosus exhibit reduced DNase I activity, and patients with myocardial infarction exhibit increased DNase I activity. So the assay of DNase I is of high importance. A colorimetric assay is described here for the determination of the activity of DNase. It is based on strand scission of dsDNA as catalyzed by DNase I. The products of digestion (nucleoside monophosphates) can better stabilize citrate capped AuNPs than dsDNA. In the absence of DNase I, the AuNPs aggregate in presence of NaCl and then display a blue color. In the presence of the analyte (DNase I), AuNPs do not aggregate but rather remain dispersed and display a red color. These findings were exploited to design a DNase I activity assay that is based on the measurement of ratio of absorbances at 520 nm (red) to 650 nm (blue). The detection limit for DNase I activity is found to be 7.1 U?L?1. In our perception, this assay has a large potential with respect to diagnoses of DNase I activity-related diseases and in drug screening.
Graphical Abstract A method is described for the determination of the activity of DNase I. It is based on the capability of nucleoside monophosphates (dNMPs; formed by DNase-catalyzed scission of dsDNA) to stabilize red gold NPs against NaCl-induced aggregation. AuNPs stabilized with dsDNA, in contrast, readily aggregate in presence of NaCl to form blue clusters.
This review (with (318) refs) describes progress made in the design and synthesis of morphologically different metal oxide nanoparticles made from iron, manganese, titanium, copper, zinc, zirconium, cobalt, nickel, tungsten, silver, and vanadium. It also covers respective composites and their function and application in the field of electrochemical and photoelectrochemical sensing of chemical and biochemical species. The proper incorporation of chemical functionalities into these nanomaterials warrants effective detection of target molecules including DNA hybridization and sensing of DNA or the formation of antigen/antibody complexes. Significant data are summarized in tables. The review concludes with a discussion or current challenge and future perspectives.
The authors report on an aptamer-based electrochemical assay for the insecticide acetamiprid. It is based on the target-induced release of the redox probe Methylene Blue (MB) from the dsDNA formed between aptamer and complementary strand (Apt/CS), exonuclease I (Exo I) and silica nanoparticles coated with streptavidin (SiNP-Streptavidin). MB is detected electrochemically using an unmodified gold electrode. In the presence of acetamiprid, MB is released from the Apt/CS dsDNA and accumulated in the close environment of the gold electrode. This results in a strong electrochemical signal for MB at fairly low working voltage of typically ?0.27 V. In the absence of target, however, the SiNP-streptavidin conjugate modified MB-dsDNA remains intact. Hence, the electrochemical signal remains weak. The method displays high selectivity for acetamiprid and a limit of detection as low as 153 pM. The assay was successfully applied to the determination of acetamiprid in (spiked) water and serum samples, with LODs of 161 and 209 pM, respectively.
Graphical abstract Schematic illustration of acetamiprid detection based on electrochemical assay. In the absence of acetamiprid, MB-dsDNA-modified SiNP-Streptavidin complex is intact and redox probe (MB) does not exist in the environment of electrode, resulting in a weak electrochemical signal (a). In the presence of target, Apt binds to acetamiprid and CS and MB leave the SiNP-Streptavidin. Exo I digests CS. So, a huge amount of MB is present in the environment of electrode and a strong electrochemical signal is observed (b).
MicroRNAs (miRNAs) play a considerable role in cancer occurrence and development, and have been identified as promising noninvasive biomarkers. The authors describe a voltammetric method for the determination of the cancer biomarker microRNA-21 (miRNA). It is based on a combination of a universal DNA signal transducer and isothermal target recycling amplification. A hairpin capture probe is bound to the target miRNA to form a duplex structure and to create a toehold in the transducer for initiating the target recycling amplification reaction. In contrast to traditional capture probes, a mismatched site is introduced to improve its ability to capture the target. In order to reduce the complex design procedures of the sequence and widen the applicability of this method, a signal transducer is introduced. Under optimal conditions, response to target miRNA is linear in the 0.5 to 2000 pM concentration range, with a 56 fM. detection limit (at an S/N ratio of 3). In order to characterize the process of target recycling and the stepwise modification of the electrode, real-time fluorescence, agarose gel electrophoresis, cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry were used. The results indicate that this isothermal target recycling amplification results in an electrochemical biosensing scheme with wide potential for sensing other bioanalytes.
Graphical abstract Schematic illustration of the electrochemical biosensing platform for miRNA-21 detection based on isothermal target recycling amplification and DNA signal transducer triggered strategy.
It is known that the binding of certain proteins to small molecules in ssDNA/small-molecule chimeras protects the conjugated ssDNA from degradation by exonuclease I (Exo I). This has resulted in numerous methods to specifically detect the interaction between small molecules and proteins. We are presenting here an approach that utilizes the terminal protection strategy in combination with the formation of ssDNA-templated silver nanoclusters (AgNCs), thereby providing a fluorometric tool for the detection of such interactions. A C-rich ssDNA (type 5′-CCCCACCCCT-3′) was labelled with biotin at the 3′ end. In the absence of streptavidin (SA), the biotinylated ssDNA is hydrolyzed in the 3′ to 5′ direction by Exo I to form mononucleotides. The formation of the AgNCs is prevented due to the lack of the DNA scaffold, and this results in weak fluorescence. Conversely, in the presence of SA, the specific binding of SA to the biotinylated ssDNA protects the ssDNA from digestion. As a result, fluorescent AgNCs are being formed. Fluorescence is measured at excitation/emission wavelengths of 625/705 nm. The calibration plot for SA is linear in the 6 to 600 nM concentration range, with a 2.6 nM detection limit. The assay is simple, sensitive and affordable. Conceivably, the method may also be used to detect the binding of other small molecules to proteins.
Graphical abstract A fluorescent sensing platform for small molecule-protein interaction assay has been developed based on terminal protection strategy and ssDNA-templated silver nanoclusters (AgNCs).
The authors describe a method for DNA target recognition and signal amplification that is based on the target-induced formation of a three way junction. The subsequent assembly of two DNA probes releases the inhibitory strand and triggers a downstream strand displacement amplification. This causes the formation of a G-rich single sequence that binds to a hemin monomer with its peroxidase-mimicking properties. The resulting peroxidase (POx) activity is quantified by using H2O2 and TMB as the substrate. In the presence of an inhibitor, in contrast, the POx-like activity is strongly reduced. This forms the basis for a highly sensitive DNA assay. It has a 0.8 pM detection limit when operated at a wavelength of 450 nm and was applied to the isothermal determination of target DNA with high selectivity.
Graphical abstract Schematic of the assay: Introduction of target results in the formation of a three way junction. The subsequent assembly of two probes releases the inhibitory strand and triggers a downstream strand displacement amplification, generating amount of G-rich single sequence which causes peroxidase-mimicking activity on binding to a hemin monomer.
The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.
Graphical abstract Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.
The authors describe a colorimetric method for the determination of DNA based on the deaggregation of gold nanoparticles (AuNPs) induced by exonuclease III (Exo III). DNA amplification is accomplished by Exo III to generate large quantities of the residual DNA. Residual DNA tethers onto the surfaces of AuNPs which prevents their aggregation. Hence, the color of the solution is red. However, in the absence of DNA, salt-induced aggregation is not prevented, and the bluish-purple color of the aggregated AuNPs is observed. The ratio of absorbances at 525 and 625 nm increases up to 150 nM DNA concentrations, and the LOD is as low as 3.0 nM. It is shown that the presence of 300 nM concentrations of random DNA (with a mass up to 10-fold that of target DNA) does not interfere. The method was successfully applied to the analysis of DNA in spiked serum samples. The method is simple, reliable, and does not require complicated amplification steps and expensive instrumentation.
Graphical abstract Schematic of a sensing strategy for DNA detection by exonuclease III-induced deaggregation of gold nanoparticles. DNA concentrations as low as 3 nM can be detected via colorimetric monitoring of the color change from red to purple-blue.
