High sensitive and label-free colorimetric DNA detection based on nicking endonuclease-assisted activation of DNAzymes

The Key Lab of Analysis and Detection Technology for Food Safety of the MOE, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002, PR China.
Talanta (Impact Factor: 3.55). 07/2011; 85(1):91-6. DOI: 10.1016/j.talanta.2011.03.042
Source: PubMed


Horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) attracts growing interest as an amplifying label for biorecognition and biosensing events, especially for DNA detection. However, in the traditional designs, one target molecule can only generate one HRP-DNAzyme, which limits the signal enhancement and thus its sensitivity. In this article, we propose an amplified and label-free colorimetric DNA detection strategy based on nicking endonuclease (NEase)-assisted activation of HRP-DNAzymes (NEAA-DNAzymes). This new strategy relies on the hairpin-DNAzyme probe and NEase-assisted target recycling. In the hairpin-DNAzyme probe, the HRP-DNAzyme sequence is protected in a "caged" inactive structure, whereas the loop region includes the target complementary sequence. Upon hybridization with target, the beacon is opened, resulting in the activation of the HRP-DNAzyme. Meanwhile, upon formation of the duplex, the NEase recognizes a specific nucleotide sequence and cleaves the hairpin-DNAzyme probe into two fragments. After nicking, the fragments of the hairpin-DNAzyme probe spontaneously dissociate from the target DNA. Amplification is accomplished by another hairpin-DNAzyme probe hybridizing to the released intact target to continue the strand-scission cycle, which results in activation of numerous DNAzymes. The activated HRP-DNAzymes generate colorimetric or chemiluminescence readout signals, thus providing the amplified detection of DNA. The detection limit of the colorimetric method is 10 pmol/L, which are three orders of magnitude lower than that without NEase. In addition, the detection limit of the chemiluminescence method is 0.2 pmol/L. Meanwhile, this strategy also exhibits high discrimination ability even against single-base mismatch.

22 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel label-free and sensitive fluorescent aptasensor for the detection of potassium ion (K(+)) was developed based on the horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme). In this work, we selected a K(+)-stabilized single stranded DNA (ssDNA) with G-rich sequence as the recognition element. In the presence of K(+), the G-rich DNA folded into the G-quadruplex structure, and then hemin can bind to the G-quadruplex structure as a co-factor and form HRP-DNAzyme. 3-(p-Hydroxyphenyl)-propanoic acid (HPPA) can be oxidized by H(2)O(2) into a fluorescent product in the presence of DNAzyme. The fluorescence intensity of the HPPA oxidative product increased with the K(+) concentration. Under the optimal conditions, the fluorescence intensity was linearly related to the logarithm of K(+) concentration in the range of 2.5 μM to 5mM. Other metal ions, such as Na(+), Li(+), NH(4)(+), Mg(2+) and Ca(2+) caused no notable interference on the detection of K(+).
    Talanta 01/2012; 89:57-62. DOI:10.1016/j.talanta.2011.11.056 · 3.55 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Specific and sensitive detection of proteins in biotechnological applications and medical diagnostics is one of the most important goals for the scientific community. In this study, a new protein assay is developed on the basis of hairpin probe and nicking enzyme assisted signal amplification strategy. The metastable state hairpin probe with short loop and long stem is designed to contain a protein aptamer for target recognition. A short Black Hole Quencher (BHQ)-quenching fluorescence DNA probe (BQF probe) carrying the recognition sequence and cleavage site for the nicking enzyme is employed for fluorescence detection. Introduction of target protein into the assay leads to the formation change of hairpin probe from hairpin shape to open form, thus faciliating the hybridization between the hairpin probe and BQF probe. The fluorescence signal is amplified through continuous enzyme cleavage. Thrombin is used as model analyte in the current proof-of-concept experiments. This method can detect thrombin specifically with a detection limit as low as 100 pM. Additionally, the proposed protein detection strategy can achieve separation-free measurement, thus eliminating the washing steps. Moreover, it is potentially universal because hairpin probe can be easily designed for other proteins by changing the corresponding aptamer sequence.
    Analytical Chemistry 04/2012; 84(8):3507-13. DOI:10.1021/ac2026783 · 5.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A novel three-way junction DNAzyme based probe has been designed for the colorimetric sensing of target DNA. Specifically, a DNAzyme-linked hairpin DNA is used as a signal probe. In the presence of target DNA, the signal probe, assistant probe and target DNA can hybridize with each other, resulting in the formation of a three-way junction DNA. At the same time, the signal probe is opened and the DNAzyme sequence in the signal probe is dehybridized. Subsequently, in the presence of hemin, the DNAzyme sequence forms a G-quadruplex-hemin complex, which catalyzes oxidation of 2, 2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by H(2)O(2) to the colored ABTS(.-)radical. The significant color changes can be distinguished visually. By the combination of the hairpin probe and the three-way junction DNA probe, the proposed sensor exhibits high recognition property for single-nucleotide polymorphisms (SNPs). This sensor allows the detection of target DNA at a concentration as low as 0.25nmol L(-1). The proposed sensor is easy to fabricate, which avoids the tedious and expensive labeling procedures, and exhibits high selectivity against single-base mismatched DNA.
    Biosensors & Bioelectronics 09/2012; 41(1). DOI:10.1016/j.bios.2012.08.056 · 6.41 Impact Factor
Show more