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.
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
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.
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.
The authors describe a novel assay for the detection of methylated DNA site. Rolling circle amplification and CdSe/ZnS quantum dots with high fluorescence efficiency are applied in this method. The CdSe/ZnS quantum dots act as electron donors, and hemin and oxygen (derived from hydrogen peroxide act as acceptors in photoinduced electron transfer. The assay, best performed at excitation/emission peaks of 450/620 nm, is sensitive and specific. Fluorometric response is linear in the 1 pM to 100 nM DNA concentration range, and the lowest detectable concentration of methylated DNA is 142 fM (S/N =?3). The method is capable of recognizing 0.01% methylated DNA in a mixture of methylated/unmethylated DNA.
Graphical abstract A novel method for methylated sites detection in DNA is established. Rolling circle amplification and photoinduced electron transfer. CdSe/ZnS quantum dots with high fluorescence efficiency act as the electron donor, while G-quadruplex/hemin and hydrogen peroxide derived oxygen act as electron acceptor. It presents a linear response towards 1 pM to 100 nM methylated DNA with a correlation coefficient of 0.9968, and the lowest detectable concentration of methylated DNA was 142 fM, with selectivity significantly superior to other methods.
A highly selective and sensitive aptasensor is described for voltammetric determination of the pesticide chlorpyrifos (CPS). The sensor was constructed by modifying a glassy carbon electrode (GCE) with gold nanorods and a polymer that was molecularly imprinted with an aptamer against CPS. This results in double specific recognition. Under optimal conditions and a working potential as low as 0.22 V (vs. Ag/AgCl), the nanotools has a dynamic range that covers the 1.0 fM - 0.4 pM CPS concentration range, and the detection limit is 0.35 fM. This is lower than any of the previously reported methods. This MIP-aptasensor is selective over structural analogs, stable, and adequately reproducible. It was successfully applied to the determination of CPS in spiked food samples.
Graphical abstract Impedimetric detection of Chlorpyrifos by using a Fe(CN)63?/4- probe based on double recognition of aptamer-molecular imprinted polymer onto a glassy carbon electrode modified with gold nanorod nanocomposite. The incubation with Chlorpyrifos lead to an increase of electron transfer resistance.
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.
The 16 kDa heat shock protein (16 kDa HSP) against Mycobacterium tuberculosis (MT), expressed during the growth phase of MT, is a potential target in diagnostic tests for tuberculosis (TB). We describe here a method for impedimetric determination of the antigen by using a nanogapped dielectric surface consisting of a silver support coated with a thin finger-shaped coating made from zinc oxide and gold and patterned through a lift-off process. The electrode was characterized by scanning electron microscopy, field emission scanning electron microscopy, atomic force microscopy, and energy-dispersive X-ray spectroscopy. Surface chemical functionalization and immobilization of antibody against the 16 kDa HSP was evidenced by FTIR. In order to improve the detection limit, the antigen was conjugated to 10 nm gold nanoparticles. The resulting biosensor is capable of detecting the 16 kDa HSP in concentrations as low as 100 fM. The method covers a wide analytical range that extends from 100 fM to 1 nM.
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.
An amperometric aptasensor is reported for the electrochemical determination of the epithelial cell adhesion molecule (EpCAM). It is based on a combination of EpCAM-driven toehold-mediated DNA recycling amplification, the specific recognition of EpCAM aptamer, and its binding to EpCAM. Hairpin probe 1 (Hp1) with a toehold region was modified with a 5′-thiol group (5’-SH) and self-assembled onto the surface of a gold electrode. Upon addition of EpCAM, the probe A (a 15-mer) is liberated from the aptamer/probe A complex and then hybridizes with the toehold domain of Hp1. This results in the exposure of another toehold for further hybridizing with hairpin probe 2 (Hp2) to displace probe A in the presence of Hp2 that was labeled with the electrochemical probe Methylene Blue (MB). Subsequently, liberated probe A is hybridized again with another Hp1 to start the next round of DNA recycling amplification by reusing probe A. This leads to the formation of plenty of MB-labeled DNA strands on the electrode surface and generates an amplified current. This 1:N probe-response amplification results in ultrasensitive and specific detection of EpCAM, with a 20 pg·mL?1 detection limit. The electrode is highly stable and regenerable. It was successfully applied to the determination of EpCAM in spiked human serum, urine and saliva, and thus provides a promising tool for early clinical diagnosis.
Graphical abstract Schematic illustration of the electrochemical detection for EpCAM. The method is based on aptamer-based recognition and EpCAM-driven toehold-mediated DNA recycling amplification. Hp1: Hairpin probe 1; Hp2: Hairpin probe 2; MB: Methylene blue; MCH: 6-Mercapto-1-hexanol; EpCAM: Epithelial cell adhesion molecule.
The work describes a hybrid electrochemical sensor for highly sensitive detection of the anesthetic lidocaine (LID). Porous carbon (PC) was synthesized from an isoreticular metal-organic framework-8 (IRMOF-8) and drop cast onto a glassy carbon electrode (GCE). A layer of a molecularly imprinted polymer (MIP) layer was then fabricated in situ on the modified GCE by electro-polymerization, with LID acting as the template and resorcinol as the functional monomer. Hexacyanoferrate is used as an electrochemical probe. The electrical signal (typically acquired at 0.335 V vs. SCE) increases linearly in the 0.2 pM to 8 nM LID concentration range, with a remarkable 67 fM detection limit (at an S/N ratio of 3). The sensor is stable and selective. Eventually, rapid and accurate detection of LID in spiked real samples was successfully realized.
The authors describe an electrochemical sensing strategy for highly sensitive and specific detection of target (analyte) DNA based on an amplification scheme mediated by a multicomponent nucleic acid enzyme (MNAzyme). MNAzymes were formed by multicomponent complexes which produce amplified “output” signals in response to specific “input” signal. In the presence of target nucleic acid, multiple partial enzymes (partzymes) oligonucleotides are assembled to form active MNAzymes. These can cleave H0 substrate into two pieces, thereby releasing the activated MNAzyme to undergo an additional cycle of amplification. Here, the two pieces contain a biotin-tagged sequence and a byproduct. The biotin-tagged sequences are specifically captured by the detection probes immobilized on the gold electrode. By employing streptavidinylated alkaline phosphatase as an enzyme label, an electrochemical signal is obtained. The electrode, if operated at a working potential of 0.25 V (vs. Ag/AgCl) in solution of pH 7.5, covers the 100 pM to 0.25 μM DNA concentration range, with a 79 pM detection limit. In our perception, the strategy introduced here has a wider potential in that it may be applied to molecular diagnostics and pathogen detection.
Graphical abstract An electrochemical strategy for sequence-specific DNA detection based on multicomponent nucleic acid enzyme (MNAzyme) -mediated signal amplification.
Intercalating fluorescent probes are widely used to visualize DNA in studies on DNA-protein interactions. Some require the presence of adenosine triphosphate (ATP). We have investigated the mechanical properties of DNA stained with the fluorescent intercalating dyes YOYO-1 and YOYO-3 as a function of ATP concentrations (up to 2 mM) by stretching single molecules in nanofluidic channels with a channel cross-section as small as roughly 100?×?100 nm2. The presence of ATP reduces the length of the DNA by up to 11 %. On the other hand, negligible effects are found if DNA is visualized with the minor groove-binding probe 4′,6-diamidino-2-phenylindole. The apparent drop in extension under nanoconfinement is attributed to an interaction of the dye and ATP, and the resulting expulsion of YOYO-1 from the double helix.
Graphical Abstract Nanochannel-stretched DNA (48.5 kbp) stained with YOYO-1 is sensitive to ATP concentration in buffer. Nanochannels with a cross-section of 80?×?80 nm2 were used to stretch DNA.
Gold nanoparticles (AuNPs) were electrodeposited on the surface of a glassy carbon electrode (GCE) and then treated with a mixture of a thiolated DNA sequence (p-63; with high affinity for bisphenol A) and free bisphenol A (BPÀ). Pyrrole was then electropolymerizaed on the surface of the GCE to entrap the BPA@p-63 complex. BPA is then extracted with acetic acid solution to obtain MIP cavities where the embedded DNA sequence acts as the binding site for BPA. Scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry were employed to characterize the surface of the modified GCE. Under the optimum conditions, the assay has a dynamic range that covers the 0.5 fM to 5 pM BPA concentration range and an 80 aM detection limit. It was applied to the quantitation of BPA in (spiked) milk, milk powder and water samples and gave acceptable recoveries.
Graphical Abstract Schematic of the procedure for aptamer-based detection of BPA using unique features of the aptamer-based modified electrodes and MIP-based sensors. This assay has high sensitivity and good selectivity. It can presumably be transferred to other detection schemes for small molecules.
Anabolic androgenic steroids (AAS) are frequently abused in human and animal sports as performance-enhancing drugs, and consequently their use is controlled by international sports authorities. Testosterone is one of the most frequently used AAS, and therefore the accurate determination of its levels in biological fluids is very important. The authors describe the selection of testosterone-binding aptamers performed using a classic SELEX approach with the target immobilized on magnetic beads. Counter selections with structurally similar steroids were implemented at different stages. Pools from different selection rounds were sequenced with Next Generation Sequencing and ten aptamer candidates were selected for further characterization. Low nanomolar range dissociation constants were calculated by a bead-based PCR assay and verified by microscale thermophoresis. Future work will focus on the development of aptamer-based platforms for the sensitive detection of testosterone in biological samples and the validation of these assays for the rapid screening of suspicious samples.
Graphical abstract The selection of testosterone-binding aptamers is described via classic SELEX using the target immobilized on magnetic beads combined with Next Generation Sequencing. The process let to the identification of several unique aptamer candidates which were characterized and their binding to testosterone was evaluated.
MicroRNAs are endogenous noncoding RNAs that play critical roles in biological processes and can be considered as molecular markers for early diagnosis and pathogenesis of diseases. The authors describe a highly sensitive electrochemical biosensor for microRNA that is based on the use of tetrahedral DNA nanostructure probes and guanine nanowire amplification. The DNA tetrahedral probe is self-assembled on a gold electrode and enhances reactivity, accessibility, and molecular recognition efficiency. Combined with the tetrahedral probe, the guanine nanowire amplifies the signal and improves the analytical performance of the biosensor. Operated best at a voltage of typically 150 mV (vs. Ag/AgCl), the sensor has a linear response to the logarithmic microRNA concentration in the 500 f. to 10 nM range, with a 176 f. detection limit. It is highly selective and can be applied to real samples. It is concluded that this strategy has a good potential with respect to the determination of microRNA in clinical diagnosis and in biological research.
Graphical abstract Schematic of a tetrahedral DNA nanostructure-based amperometric biosensor coupled to guanine nanowire amplification for analysis of microRNA-21. This strategy is highly selective and also performs well for the detection of microRNA levels of breast cancer patients.
Electrochemical DNA sensors represent a simple, accurate and economical platform for DNA detection. Gold nanoparticles are known to be efficient labels in electrochemical sensors and to be viable materials to modify the surface of electrodes thereby to enhance the detection limit of the sensor. For surface modification, gold nanoparticles are used in combination with nanomaterials like graphene, graphene oxide, or carbon nanotubes to improve electrochemical performance in general. This review (with 116 refs.) mainly covers the advances made in recent years in the use of gold nanoparticles in DNA sensing. It is divided into the following main sections: (a) An introduction covers aspects of electrochemical sensing of DNA and of appropriate nanomaterials in general. (b) The use of gold nanoparticles in DNA is specifically addressed next, with subsections on AuNPs acting as electrochemical labels, electron transfer mediators, signal amplifiers, carriers of electroactive molecules, catalysts, immobilization platforms, on silver enhancement strategies, on AuNPs modified with carbonaceous materials (such as graphenes and nanotubes), and on multiple amplification schemes. The review concludes with a discussion of current challenges and trends in terms of highly sensitive DNA based sensing using AuNPs.
Graphical abstract The review describes the state of the art in the use of gold nanoparticles in the electrochemical DNA sensors and contains sections on the use of AuNPs as labels, signal amplifiers, carriers of electroactive molecules, catalyst, immobilization platform, and on silver enhancement and multiple amplification strategies.
The authors describe an electrochemical method for the determination of the single-stranded DNA (ssDNA) oligonucleotide with a sequence derived from the genom of hepatitis B virus (HBV). It is making use of circular strand displacement (CSD) and rolling circle amplification (RCA) strategies mediated by a molecular beacon (MB). This ssDNA hybridizes with the loop portion of the MB immobilized on the surface of a gold electrode, while primer DNA also hybridizes with the rest of partial DNA sequences of MB. This triggers the MB-mediated CSD. The RCA is then initiated to produce a long DNA strand with multiple tandem-repeat sequences, and this results in a significant increase of the differential pulse voltammetric response of the electrochemical probe Methylene Blue at a rather low working potential of ?0.24 V (vs. Ag/AgCl). Under optimal experimental conditions, the assay displays an ultrahigh sensitivity (with a 2.6 aM detection limit) and excellent selectivity. Response is linear in the 10 to 700 aM DNA concentration range.
Graphical abstract Schematic of a voltammetric method for the determination of attomolar levels of target DNA. It is based on molecular beacon mediated circular strand displacement and rolling circle amplification strategies. Under optimal experimental conditions, the assay displays an ultrahigh sensitivity with a 2.6 aM detection limit and excellent selectivity.
An aptamer based assay is described for the colorimetric detection of adenosine. The presence of adenosine triggers the deformation of hairpin DNA oligonucleotide (HP1) containing adenosine aptamer and then hybridizes another unlabeled hairpin DNA oligonucleotide (HP2). This leads to the formation of a double strand with a blunt 3′ terminal. After exonuclease III (Exo III)-assisted degradation, the guanine-rich strand (GRS) is released from HP2. Hence, the adenosine-HP1 complex is released to the solution where it can hybridize another HP2 and initiate many cycles of the digestion reaction with the assistance of Exo III. This leads to the generation of a large number of GRS strands after multiple cycles. The GRS stabilize the red AuNPs against aggregation in the presence of potassium ions. If, however, GRS forms a G-quadruplex, it loses its ability to protect gold nanoparticles (AuNPs) from salt-induced AuNP aggregation. Therefore, the color of the solution changes from red to blue which can be visually observed. This colorimetric assay has a 0.13 nM detection limit and a wide linear range that extends from 5 nM to 1 μM.
Graphical abstract Schematic presentation of a colorimetric aptamer biosensor for adenosine detection based on DNA cycling amplification and salt-induced aggregation of gold nanoparticles.
Gold nanoclusters (AuNCs) protected with a bovine serum albumin (BSA) coating are known to emit red fluorescence (peaking at 650 nm) on photoexcitation with ultraviolet light (365 nm). On addition of Cu(II) ions, fluorescence is quenched because Cu(II) complexes certain amino acid units in the BSA chain. Fluorescence is, however, restored if pyrophosphate (PPi) is added because it will chelate Cu(II) and remove it from the BSA coating on the AuNCs. Because PPi is involved in the function of telomerase, the BSA@AuNCs loaded with Cu(II) can act as a fluorescent probe for determination of the activity of telomerase. A fluorescent assay was worked out for telomerase that is highly sensitive and has a wide linear range (10 nU to 10 fM per mL). The fluorescent probe was applied to the determination of telomerase activity in cervix carcinoma cells via imaging. It is shown that tumor cells can be well distinguished from normal cells by monitoring the differences in intracellular telomerase activity.
Graphical abstract Gold nanoclusters (AuNCs) protected by bovine serum albumin (BSA) and displaying red photoluminescence were prepared as fluorescent probe for the determination of telomerase activity and used for imaging of cervix carcinoma (HeLa) cells.
