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.
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.
A fluorometric ATP assay is described that makes use of carbon dots and graphene oxide along with toehold-mediated strand displacement reaction. In the absence of target, the fluorescence of carbon dots (with excitation/emission maxima at 360/447 nm) is strong and in the “on” state, because the signal probe hybridizes with the aptamer strand and cannot combine with graphene oxide. In the presence of ATP, it will bind to the aptamer and induce a strand displacement reaction. Consequently, the signal probe is released, the sensing strategy will change into the “off” state with the addition of graphene oxide. This aptasensor exhibits selective and sensitive response to ATP and has a 3.3 nM detection limit.
Graphical abstract Schematic of signal amplification by strand displacement in a carbon dot based fluorometric assay for ATP. This strategy exhibits high sensitivity and selectivity with a detection limit as low as 3.3 nM.
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
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.
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.
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.
An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2? causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs.
Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.
A sandwich-type electrochemical DNA sensor is described for the detection of oligonucleotides typical for MECP2 gene mutations. Palladium nanoparticles (PdNPs) and platinum nanoparticles (PtNPs) were used to synthesize flower-like PdPt nanodendrites (NDs) by a one-pot method. The PdPt NDs possess a high specific surface area and excellent catalytic capabilities. They served as the carrier for the signal DNA probe (SP) and simultaneously catalyze the reduction of hydrogen peroxide (H2O2). The PdPt NDs were modified with melamine, and this results in the formation of a PdPt-melamine network through stable interactions between the PdPt NDs and the three amino groups of each melamine molecule. The network exhibits excellent catalytic ability in enhancing the current signal response in the voltammetric detection of MECP2 gene mutation, best measured at ?0.4 V vs. SCE and using H2O2 as the electrochemical probe. In addition, gold nanoflowers were electrodeposited on the electrode interface in order to accelerate electron transfer and to capture the capture probe. The sensor is stable and can detect MECP2 gene mutations in the 1 fmol·L?1 to 1 nmol·L?1 concentration range, with a 0.33 fmol·L?1 lower detection limit at an S/N ratio of 3.
Graphical abstract Schematic presentation of electrodes for the determination of the X-linked gene methyl-CpG-binding protein 2 (MECP2). The sensor is based on the electrooxidation of added H2O2 by using the melamine modified palladium platinum bimetal nanodendrites as network signal amplification strategy. This versatile platform expands studies on the detection of monogenic disease.
The authors describe a method for signal amplification in electrochemical aptasensors. It is based on the induction of an increased electrochemical current by the aptamer captured on a glassy carbon electrode (GCE). The phosphate groups on the aptamer backbone are brought to reaction with added molybdate to form a redox-active molybdophosphate precipitate on the surface of the GCE that generates a strong electrochemical current. To further enhance sensitivity, gold nanorods (GNRs) were selected as a support for the immobilization of aptamers. The aptasensor was applied to the determination of the cancer biomarker carcinoembryonic antigen (CEA) in a sandwich format. Antibody against CEA, CEA (antigen) and GNRs modified with CEA aptamer were sequentially captured on the GCE. The resulting aptasensor, best operated at a voltage as low as 0.18 V vs. Ag/AgCl, is highly sensitive and has a wide linear range that extends from 0.1 pg·mL?1 to 10 ng·mL?1 of CEA. This amplification strategy uses an aptamer as both the recognition probe and signal probe and therefore simplifies signal transduction. Conceivably, this detection scheme may be adapted to numerous other electrochemical bioassays if respective antibodies and aptamers are available.
Graphical abstract Schematic presentation of an electrochemical aptasensor based on aptamer induced electrochemical current for the detection of cancer biomarker carcinoembryonic antigen (CEA). Gold nanorods (GNR) are chosen for the immobilization of aptamers to increase the loading of aptamers.
The authors describe a voltammetric immunosensor with antibody immobilized on a glassy carbon electrode (GCE) modified with N-doped graphene (N-GS), electrodeposited gold nanoparticles (AuNPs) and chitosan (Chit). The preparation is simple and the thickness of the electrodeposited films can be well controlled. Due to the specific advantages of N-GS, AuNPs and Chit, the electrode has a large specific surface, improved conductivity, high stability. A new label-free immunosensor for the model antigen (alpha fetoprotein, AFP) detection was then designed by employing N-GS-AuNP-Chit as the antibody immobilization and signal amplification platform. Differential pulse voltammetry and electrochemical impedance spectroscopy were used for the characterization of the stepwise assembly process. Under the optimized conditions, at a typical working potential of +0.20 V (vs. SCE), and by using hexacyanoferrate as an electrochemical probe, the immunosensor has a detection limit as low as 1.6 pg mL?1 and a linear analytical range that extends from 5 pg mL?1 to 50 ng mL?1. AFP was quantified in spiked human serum samples with acceptable precision.
Graphical Abstract Schematic of sensitive and effective label-free electrochemical immunosensor for the detection of AFP based on N-GS-AuNP-Chit as signal amplification matrix.
Caspases, especially caspase-3, play a critical role in the intrinsic and extrinsic pathways of apoptosis. In addition, caspase-3 is involved in mental disorders like Alzheimer disease. Any up and down regulation of caspase-3 activity may cause cancer. This review (with 58 references) summarizes recent advances in electrochemical and electrochemiluminescent quantitation of the activity of caspase-3 based on the use of nanomaterials. The nanomaterials and nanolabels are classified in three main subgroups, namely electrochemical signal amplification strategies, amplification based on modified electrodes, and the combination of both modes. The potential of various electrochemical and electrochemiluminescence bioassays is discussed, and methods to circumvent certain limitations are oresented. Finally, current trends in the detection of caspase-3 such as system integration and the application of advanced nanomaterials are discussed.
Graphical abstract The review summarizes electrochemical methods for the quantitation of caspase-3 activity based on the use of nanomaterials and of nanomaterial based labels. It contains subsections on electrochemical signal amplification strategies, amplification based on modified electrodes, and the combination of both modes.
Cardiac troponin (cTn) is a specific and sensitive biomarker for diagnosis of myocardial injury. Hence, numerous kinds of biosensors for cTn have been reported. Electrochemical methods possess inherent advantages over other kinds of sensors because they are specific, sensitive, and simple. By combining the advantages of electrochemical biosensors with those of nanomaterials, some interesting electrochemical biosensor for cTn can be obtained where the nanomaterials trigger substantial signal amplification. This review (with 101 refs.) summarizes the state of the art in electrochemical biosensing of cTn based on the use of nanomaterials. Following an introduction into the field, the use of nanomaterials in electrochemical sensing is briefly discussed. A next section covers strategies for signal amplification by using nanomaterials, with subsections on the use of nanowires, nanotubes, graphenes, and various other nanoparticles. The article concludes with a discussion of the prospects of nanomaterial-based signal amplification and on future research directions.
Graphical abstract Illustration of electrochemical biosensing of cardiac troponin (cTn) with various kinds of nanomaterials, including nanowires, nanotubes, graphene and nanoparticles, as the signal amplification modules.
MicroRNAs (miRNAs) are considered as being promising biomarkers for hematological malignancies, their aging, progression and prognosis. The authors have developed a method for the detection of miRNA-155 by using surface plasmon resonance (SPR) imaging coupled to a nucleic acid-based amplification strategy using gold nanoparticles (AuNPs). The target miRNA-155 is captured by surface-bound DNA probes. After hybridization, DNA-AuNP are employed for signal amplification via DNA sandwich assembly, resulting in a large increase in the SPR signal. This method can detect miRNA-155 in concentrations down to 45 pM and over dynamic that extends from 50 pM to 5 nM. The assay is highly specific and can discriminate even a single base mismatch. It also is reproducible, precise, and was successfully applied to the determination of miRNA-155 in spiked real samples where it gave recoveries in the range between 86% and 98%. This biosensor provides an alternative approach for miRNA detection in biomedical research and clinical diagnosis, which is highly effective and efficient.
Graphical abstract Schematic of a surface plasmon resonance imaging biosensor for detection of miRNA-155 using strand displacement amplification and gold nanoparticle.
The article reports an aptamer based assay for cocaine by employing graphene oxide and exonuclease III-assisted signal amplification. It is based on the following scheme and experimental steps: (1) Exo III can digest dsDNA with blunt or recessed 3-terminus, but it has limited activity to ssDNA or dsDNA with protruding 3-terminus; (2) GO can absorb the FAM-labeled ssDNA probe and quench the fluorescence of probe, while the affinity between FAM-labeled mononucleotide and GO is negligible; (3) Cocaine aptamer can be split into two flexible ssDNA pieces (Probe 1 and Probe 2) without significant perturbation of cocaine-binding abilities; (4) The triple complex consisting of Probe 1, Probe 2 and cocaine can be digested by Exo III with the similar efficiency as normal dsDNA. Cocaine aptamer is split into two flexible ssDNA pieces (Probe 2 and 3′-FAM-labeled Probe 1). Cocaine can mediate the cocaine aptamer fragments forming a triplex. The triple complex has unique characteristic with 3′-FAM-labeled blunt end at the Probe 1 and 3′-overhang end at Probe 2. If exonuclease III is added, it will catalyze the stepwise removal of fluorescein (FAM) labeled mononucleotides from the 3-hydroxy termini of the special triplex complex, resulting in liberation of cocaine. The cocaine released in this step can produce a new cleavage cycle, thereby leading to target recycling. Through such a cyclic bound-hydrolysis process, small amounts of cocaine can induce the cleavage of a large number of FAM-labeled probe 1. The cleaved FAM-labeled mononucleotides are not adsorbed on the surface of graphene oxide (GO), so a strong fluorescence signal enhancement is observed as the cocaine triggers enzymatic digestion. Under optimized conditions, the assay allows cocaine to be detected in the 1 to 500 nM concentration range with a detection limit of 0.1 nM. The method was applied to the determination of cocaine in spiked human plasma, with recoveries ranging from 92.0 to 111.8 % and RSD of <12.8 %.
An F0F1-ATPase-based aptasensor is described for the fluorometric determination of Vibrio parahaemolyticus. Chromatophores containing F0F1-ATPases were first prepared from Rhodospirillum rubrum cells. Then, an aptamer-functionalized chromatophore acts as the capture probe, and a chromatophore labeled with the pH probe fluorescein acts as the signalling probe. In the presence of V. parahaemolyticus, the rotation rate of F0F1-ATPase is decreased due to the formation of the aptamer-chromatophore complex. This leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and the fluorescence of the pH probe F1300 is accordingly decreased. The relative fluorescence varies linearly over the 15 to 1.5?×?106 cfu·mL?1Vibrio parahaemolyticus concentration range, and the limit of detection is 15 cfu·mL?1. The method was applied to analyze artificially contaminated salmon samples where it showed excellent perfomance.
Graphical abstract In this assay, aptamer functionalized chromatophores act as a capture probe, and the fluoresce in labeled chromatophores as signalling probe. The formation of aptamer-chromatophore complex leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and fluorescence intensity is accordingly decreased.
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 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.
The authors describe a fluorometric assay for ochratoxin A (OTA) that is based on the use of graphene oxide and RNase H-aided amplification. On addition of OTA, cAPT is replaced from the APT/cAPT hybridization complex and then hybridizes with RNA labeled with a fluorophore at the 5′-end. Eventually, the fluorophore is released by RNase H cleavage. As the concentration of OTA increases, more cAPTs are displaced, this leading to fluorescence enhancement (best measured at excitation/emission wavelengths of 495/515 nm). This RNase H-assisted cycle response results in strong signal amplification. The limit of detection, calculated on the basis of a signal to noise ratio of 3, is 0.08 ng·mL?1. Response is linear in the 0.08–200 ng·mL?1 OTA concentration range. The method is highly selective for OTA over ochratoxin B and aflatoxin B1. It was applied to the determination of OTA in red wine samples spiked at levels of 1, 7, and 50 ng·mL?1, and the recoveries ranged from 90.9 to 112%.
The authors describe a colorimetric method for the determination of Hg(II) ion. It is based on the color change from red to colorless as displayed by gold nanoparticle (AuNP) modified with thymine - rich DNA. Signal amplification is accomplished by free strand displacement recycling. In this strategy, Hg(II) unfolds the arch-trigger duplex due to the high affinity between Hg(II) and the thymines to form T-Hg(II)-T structures, thereby causing the release of trigger. The liberated trigger unfolds the hairpin structure of H1, and unfolded H1 further unfolds with H2. As a result, the H2 hairpin displaces trigger, and the released trigger unfolds another H1. This results in strong and enzyme-free strand displacement recycling amplification. The aggregation of DNA-AuNPs occurs in the presence of the duplex formed by hairpins H2 and H1. This results in a color change from red to colorless that can be visually observed. Under optimal conditions, the assay has a detection range over 4 orders of magnitude and a 3.4 nM detection limit. The assay is selective, sensitive, rapid and cost-effective. In our perception, it represents a useful platform for determination of Hg(II).
Graphical abstract Schematic presentation of the simple, rapid, low cost colorimetric detection of mercury(II) based on enzyme-free strand displacement amplification along with DNA-labeled AuNP.
