Signal amplification and detection via a supramolecular allosteric catalyst

The design of a supramolecular allosteric catalyst system for catalytic signal amplification and detection is presented. The catalyst was switched "on" by the introduction of an analyte that also behaves as an allosteric activator. Concentrations of Cl- ions as low as 800 nM were catalytically amplified and detected. The signal was transduced via a pH-sensitive fluorescent probe and observed visually using a laboratory, handheld UV lamp and by spectrophotometry. Furthermore, the allosteric effect was quantified using gas chromatography for a range of Cl- concentrations. This three-part detection scheme involving analyte binding, allosteric catalyst activation, and signal transduction represents a new approach to small-molecule detection.     

Enzyme-catalyzed signal amplification for electrochemical DNA detection with a PNA-modified electrode

The signal amplification technique of peptide nucleic acid (PNA)-based electrochemical DNA sensor was developed in a label-free and one-step method utilizing enzymatic catalysis. Electrochemical detection of DNA hybridization on a PNA-modified electrode is based on the change of surface charge caused by the hybridization of negatively charged DNA molecules. The negatively charged mediator, ferrocenedicarboxylic acid, cannot diffuse to the DNA hybridized electrode surface due to the charge repulsion with the hybridized DNA molecule while it can easily approach the neutral PNA-modified electrode surface without the hybridization. By employing glucose oxidase catalysis on this PNA-based electrochemical system, the oxidized mediator could be immediately reduced leading to greatly increased electrochemical signals. Using the enzymatic strategy, we successfully demonstrated its clinical utility by detecting one of the mutation sequences of the breast cancer susceptibility gene BRCA1 at a sample concentration lower than 10(-9) M. Furthermore, a single base-mismatched sample could be also discriminated from a perfectly matched sample.     

Alkaline phosphatase enzymatic signal amplification for fast, sensitive impedimetric DNA detection

Enzymatic signal amplification by the deposition of insoluble product on the electrode surface enhances impedimetric DNA detection sensitivity. This work demonstrates a method which gives the required detection sensitivity at significantly reduced enzyme reaction times, and demonstrates the capability for DNA SNP discrimination of biologically relevant sequences. This opens up the prospect of more rapid and relevant multiparameter impedimetric bioassays.     

Ultrasensitive fluorescence polarization DNA detection by target assisted exonuclease III-catalyzed signal amplification

Single stranded DNA sequences can be detected by target assisted exonuclease III-catalyzed signal amplification fluorescence polarization (TAECA-FP). The method offers an impressive detection limit of 83 aM within one hour for DNA detection and exhibits high discrimination ability even against a single base mismatch.     

Fluorescent signal amplification of carbocyanine dyes using engineered viral nanoparticles

We report enhancement in the fluorescent signal of the carbocyanine dye Cy5 by using an engineered virus as a scaffold to attach >40 Cy5 reporter molecules at fixed locations on the viral capsid. Although cyanine dye loading is often accompanied by fluorescence quenching, our results demonstrate that organized spatial distribution of Cy5 reporter molecules on the capsid obviates this commonly encountered problem. In addition, we observe energy transfer from the virus to adducted dye molecules, resulting in a highly fluorescent viral nanoparticle. We have used this enhanced fluorescence for the detection of DNA-DNA hybridization. When compared with the most often used detection methods in a microarray-based genotyping assay for Vibrio cholerae O139, these viral nanoparticles markedly increased assay sensitivity, thus demonstrating their applicability for existing DNA microarray protocols.     

Surface-initiated DNA self-assembly as an enzyme-free and nanoparticle-free strategy towards signal amplification of an electrochemical DNA sensor

Surface-initiated DNA polymerization has been employed in this work as an appealing signal amplification strategy for electrochemical DNA sensors. This strategy is especially superior in that enzymes, colloidal particles and other bulky structures are not involved in order to achieve amplified signals, and thus is highly promising in circumventing problems due to uncontrolled nucleation, adsorption, aggregation or disassembly of nanoparticles, liposomes and proteins, as well as enzyme deactivations. Our preliminary results have shown that a decrease (as compared to an amplification-free system) in detection limit by a factor greater than 300 can be easily achieved by cyclic voltammetry under still not optimized conditions, with an ability of differentiating a single base mutation.     

Ultrasensitive DNA detection based on Au nanoparticles and isothermal circular double-assisted electrochemiluminescence signal amplification

We report here an ultrasensitive DNA detection approach which combines Au NPs enhanced electrochemiluminescence (ECL) of the CdS nanocrystal (NC) film with isothermal circular amplification reaction of polymerase and nicking endonuclease (NEase). By the double-signal amplification, this approach could sensitively respond down to 5 aM DNA.     

Nicking endonuclease and target recycles signal amplification assisted quantum dots for fluorescence detection of DNA

An ultrasensitive fluorescence detection method for DNA based on nicking endonuclease (NEase) and target recycles assisted with CdTe quantum dots (QDs) is reported. In the detection system, when the target DNA is present, it hybridizes with a linker strand to from a duplex, in which the NEase recognizes specific nucleotide sequences and cleaves the linker strand. After nicking, the fragments of the linker strand spontaneously dissociate from the target DNA and another linker strand hybridizes to the target to trigger another strand-scission cycle. On the other hand, when the target was absent, no duplex is formed and no fragment of linker strand is produced. Then CdTe QDs and magnetic beads (MBs), which were all modified with DNA sequences complementary to that of the linker strands are added to the solution to detect the presence of a target DNA. The signal was generated through the difference in F?rster resonance energy transfer (FRET) between the MB and CdTe QDs. This method indicates that one target DNA leads to cleavage of hundreds of linker DNA, increasing detection sensitivity by nearly three orders of magnitude. This method should be applicable whenever there is a requirement to detect a specific DNA sequence and can also be used for multicomponent detection.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

Digital readout of target binding with attomole detection limits via enzyme amplification in femtoliter arrays

In this communication, single molecules of beta-galactosidase were captured on a 1 mm femtoliter array using biotin-streptavidin binding. The femtoliter arrays, containing 24 000 individual reaction chambers, permit digital concentration readout as the percentage of reaction vessels that successfully capture a target molecule is correlated to the bulk target concentration. This capture and readout approach should prove useful for DNA and antibody assays that utilize an enzyme label to catalyze the generation of a fluorescent signal.     

Electrochemical DNA biosensor based on MNAzyme-mediated signal amplification

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.

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Graphical abstract An electrochemical strategy for sequence-specific DNA detection based on multicomponent nucleic acid enzyme (MNAzyme) -mediated signal amplification.

     

Electrochemiluminescence detection of near single DNA molecules by using quantum dots-dendrimer nanocomposites for signal amplification

An ultrasensitive electrochemiluminescent biosensor was developed for detection of near single DNA molecules with a linear range of 7 orders of magnitude by combining the specific recognition of a molecular beacon with signal amplification of quantum dots-dendrimer nanocomposites.     

Graphene oxide-based biosensor for sensitive fluorescence detection of DNA based on exonuclease III-aided signal amplification

Based on the super fluorescence quenching efficiency of graphene oxide and exonuclease III aided signal amplification, we develop a facile, sensitive, rapid and cost-effective method for DNA detection. In the presence of target DNA, the target-probe hybridization forms a double-stranded structure and exonuclease III catalyzes the stepwise removal of mononucleotides from the blunt 3′ termini of probe, resulting in the recycling of the target DNA and signal amplification. Therefore, our proposed sensor exhibits a high sensitivity towards target DNA with a detection limit of 20 pM, which was even lower than previously reported GO-based DNA sensors without enzymatic amplification, and provides a universal sensing platform for sensitive detection of DNA.     

Activating an enzyme by an engineered coiled coil switch

We designed a de novo protein based on a circular permutant of RNaseT1, in which the enzymatic activity can be manipulated by engineered peptide binding. The circular permutant of RNaseT1 was obtained by tethering the original C- and N-termini with a GPAG linker and cleaving the molecule between Glu82 and Asn83. This mutant lacked enzymatic activity, due to the destabilization of entire protein structure. We previously reported the construction of ABC-type heterotrimeric coiled coil peptides, in which the A- and B-type peptides cannot form the folded trimeric structure without the C-type peptide. The introduction of the A- and B-type coiled coil peptides to the C- and N-termini of the circular permutant of RNaseT1, respectively, and the subsequent addition of the C-type coiled coil peptide enabled the RNaseT1 domain to refold properly, thus, restoring the enzymatic activity. The formation of the trimeric coiled coil structure should bring the cleaved sites of RNaseT1 close enough to refold the RNaseT1 domain spontaneously.     

Entropy-driven molecular switch and signal amplification for homogeneous SNPs detection

An approach was developed to rapidly and sensitively detect SNP in homogeneous solution based on an entropy-driven molecular switch and isothermal polymerase amplification reaction without addition of exogenous primers.     

Ultra-selective and sensitive DNA detection by a universal apurinic/apyrimidinic probe-based endonuclease IV signal amplification system

A novel signal amplification system that is applicable to any DNA sequence of interest has been developed by using a combination of apurinic/apyrimidinic probe and endonuclease IV. The system allows rapid, highly selective and sensitive detection of target DNA sequences at very low concentrations (10 fmol) or low abundance levels (1%).