Amplified electrochemical DNA sensor using peroxidase-like DNAzyme

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H₂O₂, which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.     

A divalent metal-dependent self-cleaving DNAzyme with a tyrosine side chain

The enzymatic incorporation of a phenol-modified 2'-deoxyuridine triphosphate gave rise to a modified DNA library that was subsequently used in an in vitro selection for ribophosphodiester-cleaving DNAzymes in the presence of divalent zinc and magnesium cations. After 11 rounds of selection, cloning and sequencing resulted in 14 distinct sequences, the most active of which was Dz11-17PheO. Dz11-17PheO self-cleaved an embedded ribocytidine with an observed rate constant of 0.20 ± 0.02 min(-1) in the presence of 10 mM Mg(2+) and 1 mM Zn(2+) at room temperature. The activity was inhibited at low concentrations of Hg(2+) cations and somewhat higher concentrations of Eu(3+) cations.     

Peptide nucleic acid stabilized perovskite nanoparticles for nucleic acid sensing

Nanostructural hybrid organic-inorganic metal halide perovskites offer a wide range of potential applications including photovoltaics, solar cells, and light emitting diodes. Up to now the surface stabilizing ligands were used solely to obtain the optimal properties of nanoparticles in terms of dimensionality and stability, however their possible additional functionality was rarely considered. In the present work, hybrid lead bromide perovskite nanoparticles (PNP) were prepared using a unique approach where a peptide nucleic acid is used as a surface ligand. Methylammonium lead bromide perovskite colloidal nanoparticles stabilized by thymine-based peptide nucleic acid monomer (PNA-M) and relevant trimer (PNA-T) were prepared exhibiting the size below 10 nm. Perovskite structure and crystallinity were verified by X-ray powder diffraction spectroscopy and high resolution transmission electron microscopy. PNP-PNA-M and PNP-PNA-T colloidal dispersions in chloroform and toluene possessed green-blue fluorescence, while Fourier-transform infrared spectroscopy (FT-IR) and quantum chemical calculations showed that the PNA coordinates to the PNP surface through the primary amine group. Additionally, the sensing ability of the PNA ligand for adenine nucleic acid was demonstrated by photoluminescence quenching via charge transfer. Furthermore, PNP thin films were effectively produced by the centrifugal casting. We envision that combining the unique, tailored structure of peptide nucleic acids and the prospective optical features of lead halide perovskite nanoparticles could expand the field of applications of such hybrids exploiting analogous ligand chemistry.     

Label-free fluorescent detection of thrombin using G-quadruplex-based DNAzyme as sensing platform

We report herein a label-free and sensitive fluorescent method for detection of thrombin using a G-quadruplex-based DNAzyme as the sensing platform. The thrombin-binding aptamer (TBA) is able to bind hemin to form the G-quadruplex-based DNAzyme, and thrombin can significantly enhance the activity of the G-quadruplex-based DNAzyme. The G-quadruplex-based DNAzyme is found to effectively catalyze the H(2)O(2)-mediated oxidation of thiamine, giving rise to fluorescence emission. This allows us to utilize the H(2)O(2)-thiamine fluorescent system for the quantitative analysis of thrombin. The assay shows a linear toward thrombin concentration in the range of 0.01-0.12 nM. The present limit of detection for thrombin is 1 pM, and the sensitivity for analyzing thrombin is improved by about 10,000-fold as compared with the reported colorimetric counterpart. The work also demonstrates that thiamine is an excellent substrate for the fluorescence assay using the G-quadruplex-based DNAzyme as the sensing platform.     

Fluorescence-enhanced nucleic acid detection: using coordination polymer colloids as a sensing platform

Coordination polymer colloids have been used as an effective fluorescent sensing platform for multiplexing nucleic acid detection capable of distinguishing complementary and mismatched target sequences for the first time.     

Advances in nucleic acid architectures for electrochemical sensing

Nucleic acid–based electrochemical sensors are ideally suited to the detection of molecular targets for which enzymatic detection or direct electrochemical oxidation – reduction reactions are not possible. Moreover, the versatility of nucleic acids in their ability to bind a great variety of target types, from small molecules to single-entity mesoscopic targets, makes them attractive receptors for the development of electrochemical biosensors. In this brief opinion piece, we discuss field advances from the past two years. We hope the works highlighted here will inspire the community to pursue creative designs enabling the detection of larger and more complex targets with a specific focus on analytical validation and translation into preclinical or clinical applications.     

Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme

G-quadruplex containing peroxidase DNAzyme is a complex of hemin and a single-stranded guanine-rich DNA (hemin-binding DNA aptamer), which is used as an attractive catalytic label for biosensing recently. Therein, the hemin-binding DNA aptamer contains four GGG repeats and can fold into a G-quadruplex structure. In this paper, we have developed a new split mode to divide the hemin-binding DNA aptamer into two parts: one possesses three GGG repeats, and another part possesses one GGG repeat, namely, the 3:1 split mode. The combination of G-quadruplex and hemin binding could be used as a sensitive probe for the identification of single nucleotide polymorphisms by giving a color signal, visible to the naked eye at room temperature. The G-quadruplex containing peroxidase DNAzyme utilizes the 3:1 split mode and can be directly used for the identification of SNPs with a detection limit in the nM range when the matching length of the probe is short enough. When the matching length of the probe is relatively long, another method adding competition sequences to the probe could also operate effectively for the identification of SNPs. The results also suggested that we could detect the signal when the mutation sample was only 5% in the total target DNA with a competition strategy.     

Homo-DNA templated chemistry and its application to nucleic acid sensing

We have investigated the homo-DNA templated Staudinger reduction of the profluorophore rhodamine azide and have applied this reaction to the detection of natural DNA with a hybrid homo-DNA/DNA molecular beacon. In this system the sensing and the reporting unit are bioorthogonal to each other which facilitates sequence design and increases fidelity.     

Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles

The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence.     

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10(-14) M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene.     

CdS quantum dots as a fluorescent sensing platform for nucleic acid detection

We demonstrate that CdS quantum dots (QDs) can be applied to fluorescence-enhanced detection of nucleic acids in a two-step protocol. In step one, a fluorescently labeled single-stranded DNA probe is adsorbed on the QDs to quench its luminescence. In step two, the hybridization of the probe with its target ssDNA produces a double-stranded DNA which detaches from the QD. This, in turn, leads to the recovery of the fluorescence of the label. The lower detection limit of the assay is as low as 1?nM. The scheme (that was applied to detect a target DNA related to the HIV) is simple and can differentiate between perfectly complementary targets and mismatches.

Single-mismatch detection using nucleic acid molecular 'light switch'

A novel nucleic acid molecular ‘light switch’ method is developed for the sensitive recognition and detection of a single-base mismatched oligonucleotides. The detection limit of oligonucleotide of perfect double stranded and that with single-base, two-base and three-base mismatched are 0.11, 0.17, 0.34 and 1.5 ng ml⁻¹, respectively. It was found that Ru(phen)₂(dppx)²⁺ (phen=1,10-phenanthroline, dppx=7,8-dimethyl-dipyridophenazine) can be used to detect and recognize the perfect double stranded oligonucleotides from mismatched and random targets by the intensity of fluorescence and temperature. This method can be used to recognize and quantitatively detect target DNA with specific sequence. The advantage of this method is that no requisites are needed to separate the coexisting random targets in the case of a mixed solution containing perfect, mismatched and random targets, which make the recognition analysis of oligonucleotide simple and fast. Moreover, it has potential in the study of dynamic process of DNA hybridization.     

Sensitive quantitative nucleic acid detection using oligonucleotide microarrays

We report a new theoretical approach to optimize the performance and quantify the results of gene expression oligonucleotide microarrays which are widely used in biomedical research. An on-array hybridization isotherm that takes into account the screened Coulomb repulsion between the assayed nucleic acid target and the layer of surface tethered oligonucleotide probes is presented. The hybridization efficiency is found as a function of the genomic target (sequence, length, and concentration), array parameters (probe sequence and length, surface probe density), and hybridization conditions (temperature and buffer ionic strength). We present simple relations for the hybridization signal maximum and the linear dynamic detection range and show explicit criteria for optimization. The approach is based on an extension of our recently published theory (Vainrub, A.; Pettitt, B. M. Phys. Rev. E 2002, 66, art. no.-041905) which we generalize here for the cases of target depletion effects and arbitrary target length.     

Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

Herein, we describe an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced indiscrimitive single-stranded DNase activity of Cas12a for the quantitative profiling of gene expression at the mRNA level and detection of proteins with high sensitivity and specificity. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows for flexible assay design. A binding-induced primer extension reaction is used to generate a predesigned CRISPR-targetable sequence as a barcode for further signal amplification. Through this dual amplification protocol, we were able to detect as low as 1 fM target nucleic acid and 100 fM target protein isothermally. The practical applicability of this assay was successfully demonstrated for the temporal profiling of interleukin-6 gene expression during allergen-mediated mast cell activation.

Herein, we develop an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced collateral cleavage activity of Cas12a for the quantitative profiling of gene expression and detection of proteins with high sensitivity and specificity.     

ECHO probes: a concept of fluorescence control for practical nucleic acid sensing

An excitonic interaction caused by the H-aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence-controlled nucleic acid sensing. This critical review points out the recent advances in exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, which have a fluorescence-labeled nucleotide in which two molecules of thiazole orange or its derivatives are linked covalently. ECHO probes show absorption shift and emission switching depending on hybridization with the target nucleic acid. The hybridization-sensitive fluorescence emission of ECHO probes and the further modification of probes have made possible a variety of practical applications, such as multicolor RNA imaging in living cells and facile detection of gene polymorphism (144 references).     

Amplified fluorescence detection of mercury(II) ions (Hg2+) using target-induced DNAzyme cascade with catalytic and molecular beacons

In this work, a fluorescent sensing strategy was developed for the detection of mercury(II) ions (Hg(2+)) in aqueous solution with excellent sensitivity and selectivity using a target-induced DNAzyme cascade with catalytic and molecular beacons (CAMB). In order to construct the biosensor, a Mg(2+)-dependent DNAzyme was elaborately designed and artificially split into two separate oligonucleotide fragments. In the presence of Hg(2+), the specific thymine-Hg(2+)-thymine (T-Hg(2+)-T) interaction induced the two fragments to produce the activated Mg(2+)-dependent DNAzyme, which would hybridize with a hairpin-structured MB substrate to form the CAMB system. Eventually, each target-induced activated DNAzyme could catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers. This would significantly enhance the sensitivity of the Hg(2+) sensing system and push the detection limit down to 0.2 nM within a 20 min assay time, much lower than those of most previously reported fluorescence assays. Owning to the strong coordination of Hg(2+) to the T-T mismatched pairs, this proposed sensing system exhibited excellent selectivity for Hg(2+) detection, even in the presence of 100 times of other interferential metal ions. Furthermore, the applicability of the biosensor for Hg(2+) detection in river water samples was demonstrated with satisfactory results. These advantages endow the sensing strategy with a great potential for the simple, rapid, sensitive, and specific detection of Hg(2+) from a wide range of real samples.     

Oligonucleotide-templated reactions for sensing nucleic acids

Oligonucleotide-templated reactions are useful for applying nucleic acid sensing. Various chemistries for oligonucleotide-templated reaction have been reported so far. Major scientific interests are focused on the development of signal amplification systems and signal generation systems. We introduce the recent advances of oligonucleotide-templated reaction in consideration of the above two points.     

Excitonic interaction: another photophysical process for fluorescence‐controlled nucleic acid sensing

An excitonic interaction caused by the H‐aggregation of fluorescent dyes is a new type of useful photophysical process for fluorescence‐controlled nucleic acid sensing. We designed a fluorescence‐labeled nucleotide in which two thiazole orange dyes were linked covalently. A DNA strand containing this fluorescence‐labeled nucleotide showed absorption at 480 nm before hybridization, whereas an absorption band at 510 nm became predominant when the DNA was hybridized with the complementary strand. The shift in the absorption bands shows the existence of an excitonic interaction between dyes in the nucleotide, and as a result, emission from the doubly thiazole orange‐labeled DNA was well controlled. This clear change in fluorescence intensity depending on hybridization is applicable to multicolor RNA imaging in living cells. © 2010 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 10: 188–196; 2010: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.201000003     

Amplified electrochemiluminescence detection of nucleic acids by hairpin probe-based isothermal amplification

Here, we present a straightforward method for isothermal amplified detection of nucleic acids. In this proof-of-concept study, a specific DNA sequence is amplified through hairpin probe-based isothermal strand-displacement polymerization reaction and then detected via a sensitive and commercially available ECL detection platform. Results show that the DNA sequence derived from the Listeria monocytogenes hly gene can be detected down to 10 pM in solution, together with correlation of the detected signal with the initial concentration of target DNA. Moreover, the designed stem-loop structured hairpin probe shows single-base variation differentiating ability. Considering the superior sensitivity and specificity, as well as the simple-to-implement features, the developed assay demonstrates a great potential of becoming a first-line tool for quantitative analysis of nucleic acids for biomedical research.