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.     

Intercalation of quantum dots as the new signal acquisition and amplification platform for sensitive electrochemiluminescent detection of microRNA

Herein, we report on the development of a simple and sensitive biosensor for electrochemiluminescent (ECL) detection of microRNAs (miRNA) based on the intercalation of doxorubicin-conjugated quantum dot nanoparticles (Dox-QDs) into the DNA/RNA hybrids as the new signal acquisition and amplification platform. The thiolated DNA capture probes are self-assembled onto gold electrodes via   the formation of Au–S bonds. The sensing surface is then incubated in a target miRNA-containing buffer solution to form the double-stranded duplexes. In this case, massive Dox-QDs can intercalate into the base pairs of the hybrid duplexes, resulting in amplified ECL emissions due to their reactions with the coreactant S₂O₈²⁻${{\text{S}}_2{\text{O}}_8}^{2-}$ and the dissolved oxygen in the detection buffer. The increase in ECL intensity proportional to the amount of target miRNA in the testing samples serves as the quantitative basis. Different from traditional QDs-based methods such as labeling and embedding, our sensor involves the employment of the intercalation of the Dox-QDs as the signal acquisition and amplification platform. The combination of the QDs intercalation amplification with the high sensitivity of the ECL technique enables us to detect miRNA down to the low femtomolar level. Moreover, our method is also coupled with acceptable selectivity in discriminating the target miRNA and against its family members as well as other interference sequence, and can monitor miRNAs from human prostate carcinoma (22Rv1) cell lysates.     

Nicking enzyme based homogeneous aptasensors for amplification detection of protein

A simple and highly sensitive homogeneous aptasensor is developed, which relies on nicking enzyme. The sensitivity of this newly proposed aptasensor is about three orders of magnitude higher than that of traditional homogeneous aptasensors. Furthermore, it is capable of detecting target protein in real samples.     

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.     

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.     

Ratiometric fluorescence detection of mercuric ion based on the nanohybrid of fluorescence carbon dots and quantum dots

A novel nanohybrid ratiometric fluorescence probe comprised of carbon dots (C-dots) and hydrophilic CdSe@ZnS quantum dots (QDs) has been developed by simply mixing the blue-emission C-dots with red-emission carboxylmethyldithiocarbamate modified CdSe@ZnS QDs (GDTC-QDs). The nanohybrid ratiometric fluorescence probe exhibits dual emissions at 436 nm and 629 nm under a single excitation wavelength. Due to the strong chelating ability of GDTC on the surface of QDs to mercuric ion (Hg²⁺), the fluorescence of the GDTC-QDs in the nanohybrid system could be selectively quenched in the presence of Hg²⁺ while the fluorescence of the C-dots remained constant, resulting in an obviously distinguishable fluorescence color evolution (from red to blue) of the nanohybrid system. The detection limit of this method was found to be as low as 0.1 μM. Furthermore, the recovery result for Hg²⁺ in real samples including tap water and lake water by this method was satisfying, suggesting its potential application for Hg²⁺ sensing.     

An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification

A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.     

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%).     

Biosensor for multiplex detection of two DNA target sequences using enzyme-functionalized Au nanoparticles as signal amplification

Multiplex electrochemical detection of two DNA target sequences in one sample using enzyme-functionalized Au nanoparticles (AuNPs) as catalytic labels for was proposed. This DNA sensor was fabricated using a “sandwich” detection strategy, involving two kinds of capture probes DNA immobilized on glassy carbon electrode (GCE), and hybridization with target DNA sequences, which further hybridized with the reporter DNA loaded on the AuNPs. The AuNP contained two kinds of DNA sequences, one was complementary to the target DNA, while the other was noncomplementary to the target. The noncomplementary sequences were linked with horseradish peroxidase (HRP) and alkaline phosphatase (ALP), respectively. Enhanced detection sensitivity was obtained where the AuNPs carriers increased the amount of enzyme molecules per hybridization. Electrochemical signals were generated from the enzymatic products produced from the substrates catalyzed by HRP and ALP. Under optimal conditions, a 33-mer sequence could be quantified over the ranges from 1.5 × 10⁻¹³ to 5.0 × 10⁻¹² M with a detection limit of 1.0 × 10⁻¹³ M using HRP-AuNP as labels, and a 33-mer sequence could be quantified over the ranges from 4.5 × 10⁻¹¹ M to 1.0 × 10⁻⁹ M with a detection limit of 1.2 × 10⁻¹¹ M using ALP-AuNP as labels.     

Ligand displacement-induced fluorescence switch of quantum dots for ultrasensitive detection of cadmium ions

This paper reports the construction of a simple CdTe quantum dots (QDs)-based sensor with 1,10-phenanthroline (Phen) as ligand, and the demonstration of a novel ligand displacement-induced fluorescence switch strategy for sensitive and selective detection of Cd²⁺ in aqueous phase. The complexation of Phen at the surface quenches the green photoluminescence (PL) of QDs dominated by a photoinduced hole transfer (PHT) mechanism. In the presence of Cd²⁺, the Phen ligands are readily detached from the surface of CdTe QDs, forming [Cd(Phen)₂(H₂O)₂]²⁺ in solution, and as a consequence the PL of CdTe QDs switches on. The detection limit for Cd²⁺ is defined as ∼0.01 nM, which is far below the maximum Cd²⁺ residue limit of drinking water allowed by the U.S. Environmental Protection Agency (EPA). Two consecutive linear ranges allow a wide determination of Cd²⁺ from 0.02 nM to 0.6 μM. Importantly, this CdTe QDs-based sensor features to distinctly discriminate between Cd²⁺ and Zn²⁺, and succeeds in real water samples. This extremely simple strategy reported here represents an attempt for the development of fluorescent sensors for ultrasensitive chemo/biodetection.     

Direct molecular detection of nucleic acids by fluorescence signal amplification

An integrated PCR-free DNA sensor, which combines a sequence-specific receptor, an optical polymeric transducer, and an intrinsic fluorescence amplification mechanism, is reported. This sensor is based on the different conformations adopted by a cationic polythiophene when electrostatically bound to ss-DNA or ds-DNA, and on the efficient and fast energy transfer between the resulting fluorescent polythiophene/ds-DNA complex and neighboring fluorophores attached to ss-DNA probes. This molecular system allows the detection of only five molecules in 3 mL of an aqueous solution, or 3 zM, in 5 min. Moreover, this work demonstrates, for the first time, the direct detection of single nucleotide polymorphisms (SNPs) from clinical samples in only a few minutes, without the need for nucleic acid amplification.     

水溶性CdTe量子点的合成及其用于荧光猝灭法测定肌红蛋白

以3-巯基丙酸为稳定剂,合成了具有特殊光学性质的水溶性CdTe量子点,其最大发射波长位于544 nm.利用荧光光谱、紫外可见光谱及圆二色光谱法系统的研究了CdTe量子点与肌红蛋白(Mb)二者结合前后体系光谱的变化,从而证实了CdTe量子点与Mb之间静电结合反应的特征.在pH 7.0的PBS缓冲液中,用CdTe量子点作为荧光探针研究了肌红蛋白与量子点的相互作用,并基于肌红蛋白对CdTe量子点有显著的荧光猝灭作用,建立了肌红蛋白的快速检测方法.在最佳实验条件下,该体系荧光强度的猝灭程度(△F)与肌红蛋白质量浓度呈良好的线性关系,线性范围为0.3～24 μg/mL,检出限为0.13 μg/mL.该方法已对合成样品中肌红蛋白进行检测,并用于人体尿样中肌红蛋白的测定.     

Immobilisation of quantum dots by bio-orthogonal PCR amplification and labelling for direct gene detection and quantitation

A sensitive and versatile detection scheme based on quantum dot immobilisation on a solid support through bio-orthogonal PCR amplification and labelling has been developed for detection and quantification of gene targets in complex DNA mixtures.     

Highly sensitive naked-eye and fluorescence "turn-on" detection of Cu(2+) using Fenton reaction assisted signal amplification

Increase of pH induced by Cu(2+)-catalyzed Fenton reaction promoted ring-opening of triazole-linked fluorescein lactone, which enabled selective "turn-on" fluorescent detection of Cu(2+), along with ultralow naked-eye detection limit down to 200 nM.     

Interactions between CdTe quantum dots and DNA revealed by capillary electrophoresis with laser‐induced fluorescence detection

Quantum dots (QDs) are one of the most promising nanomaterials, due to their size‐dependent characteristics as well as easily controllable size during the synthesis process. They are promising label material and their interaction with biomolecules is of great interest for science. In this study, CdTe QDs were synthesized under optimal conditions for 2 nm size. Characterization and verification of QDs synthesis procedure were done by fluorimetric method and with CE. Afterwards, QDs interaction with chicken genomic DNA and 500 bpDNA fragment was observed employing CE‐LIF and gel electrophoresis. Performed interaction relies on possible matching between size of QDs and major groove of the DNA, which is approximately 2.1 nm.     

Simultaneous detection of two lung cancer biomarkers using dual-color fluorescence quantum dots

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, a novel protocol for performing a simultaneous dual-protein immunoassay, i.e. two lung cancer biomarkers, carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE), based on dual-color QDs, is described. First, two capture antibodies (both with biotin tags), two antigens and two detection antibodies were mixed together and the sandwich complexes were thus formed in the homogeneous solution, and then streptavidin coated polystyrene beads were directly added into the resultant system. Bead aggregation can be made self-limiting by controlling the shaker speed during the immunoassay. A distinct transition occurs between limited and complete aggregation as a function of the shaker speed during the immunoassay. Second, dual-color QDs with emission maxima at 525 and 655 nm were added after washing and reacted with the corresponding detection antibodies. Third, the bead-QD conjugates were dissociated in the dissociation buffer and then free QDs were directly used for the fluorescence detection of CEA and NSE. The results show that CEA and NSE could be sensitively determined with a common 96-well fluorescence plate reader and with equal detection limits down to the 1.0 ng mL(-1) level. Within the calibrated amount, the protocol had excellent precision within 0.53% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual markers in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for CEA and NSE in 25 human serum samples.