Detection of microRNA using a polydopamine mediated bimetallic SERS substrate and a re-circulated enzymatic amplification system.

A surface-enhanced Raman scattering (SERS) method is described for the determination of microRNA that is associated with various forms of cancer. The substrate consists of functionalized gold-silver bimetallic structure, and the sensitivity is strongly enhanced by making use of a re-circulated enzymatic amplification system (REAS). Poly-dopamine acts as both a reductant and a protective of the substrates. It was employed to link the gold core and silver satellite. The unique "hot spots" consisting of a Au@PDA@Ag nanocomposite improve the Raman signal and sensitivity. The reductive feature of PDA can prevent the susceptible oxidation of metallic silver to maintain the high Raman activity. To improve the sensitivity of the assays, a re-circulated enzymatic amplification system was developed in which the nicking endonuclease triggers the nucleic acid reaction system to enter an amplified cycle. By integrating the bimetallic nanosubstrate and magnetic separation into the REAS, microRNA can be detected by SERS (best at the Raman band of 1586 cm-1) with a limit of detection as low as 0.2 fM. In our perception, the assay provides an exciting new avenue to study the expression of tumor genes. Thus, it holds vast promise in cancer diagnosis. Graphical abstract Schematic presentation of the SERS method based on poly-dopamine mediated bimetallic SERS substrate and re-circulated enzymatic amplification.

A single quantum dot-based biosensor for DNA point mutation assay.

Sensitive and selective detection of point mutation is essential to molecular biology research and early clinical diagnosis. Here, we demonstrate a single quantum dot (QD)-based biosensor for DNA point mutation assay. In this assay, a mutant target (G/C) remains unchanged after the endonuclease treatment, and the polymerase chain reaction (PCR) may be initiated with the assistance of primers and polymerase, generating a large number of mutant targets. The amplified mutant targets can be captured by biotinylated probes during the process of denaturation and annealing, and Cy5-dGTP may be assembled into the biotinylated probe with the catalysis of polymerase, leading to the formation of Cy5-labeled biotinylated probes. The Cy5-labeled biotinylated probes can be further assembled onto the QD surface to obtain a Cy5-DNA-QD complex, resulting in the generation of fluorescence resonance energy transfer (FRET) between the QD donor and the Cy5 receptor. The mutant targets can be quantitatively evaluated by the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy. While in the presence of wild-type targets (T/A), no Cy5-dGTP can be assembled into the biotinylated probe due to the presence of a mismatch and consequently no FRET is observed. This single QD-based biosensor exhibits high sensitivity with a detection limit of 5.3 aM (or 32 copies) and can even discriminate as low as 0.01% variant frequency from the mixture of mutant targets and wild-type ones. Importantly, this biosensor can be used for genomic analysis in human lung cancer cells, and may be further applied for an early clinical diagnosis and personalized medicine.

Quencher-free fluorescent method for homogeneously sensitive detection of microRNAs in human lung tissues.

Sensitive and accurate analysis of microRNA (miRNA) expression is imperative for understanding the biological functions of miRNAs and the early diagnosis of human cancers. Here, we develop a quencher-free fluorescent method for homogeneously sensitive detection of let-7a miRNA using the target-triggered recycling signal amplification in combination with a 2-aminopurine probe. The 2-aminopurine probe is characterized by the substitution of 2-aminopurine for adenine in the DNA strand and the quenching of 2-aminopurine fluorescence through its stacking interaction with the adjacent bases. The binding of target miRNA with the 2-aminopurine probe initiates the extension reaction in the presence of polymerase to produce the DNA duplexes. These DNA duplexes can be further cleaved by lambda exonuclease through the recycling digestion to release abundant free 2-aminopurines, leading to an enhanced fluorescence signal. The proposed method exhibits high sensitivity with a detection limit of 0.3 fmol, and it can even discriminate the single-base difference among the miRNA family members. More importantly, this method can accurately distinguish the expression of let-7a miRNA in human lung tissues between ten non small cell lung cancer (NSCLC) patients and ten healthy persons, holding a great potential for further application in early clinical diagnosis.

Homogeneous and label-free detection of microRNAs using bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification.

MicroRNAs (miRNAs) are an emerging class of biomarkers and therapeutic targets for various diseases including cancers. Here, we develop a homogeneous and label-free method for sensitive detection of let-7a miRNA based on bifunctional strand displacement amplification (SDA)-mediated hyperbranched rolling circle amplification (HRCA). The binding of target miRNA with the linear template initiates the bifunctional SDA reaction, generating two different kinds of triggers which can hybridize with the linear template to initiate new rounds of SDA reaction for the production of more and more triggers. In the meantime, the released two different kinds of triggers can function as the first and the second primers, respectively, to initiate the HRCA reaction whose products can be simply monitored by a standard fluorometer with SYBR Green I as the fluorescent indicator. The proposed method exhibits high sensitivity with a detection limit of as low as 1.8 × 10(-13) M and a large dynamic range of 5 orders of magnitude from 0.1 pM to 10 nM, and it can even discriminate the single-base difference among the miRNA family members. Moreover, this method can be used to analyze the total RNA samples from the human lung tissues and might be further applied for sensitive detection of various proteins, small molecules, and metal ions in combination with specific aptamers.

Functional nucleic acid-based sensors for heavy metal ion assays.

Heavy metal contaminants such as lead ions (Pb(2+)), mercury ions (Hg(2+)) and silver ions (Ag(+)) can cause significant harm to humans and generate enduring bioaccumulation in ecological systems. Even though a variety of methods have been developed for Pb(2+), Hg(2+) and Ag(+) assays, most of them are usually laborious and time-consuming with poor sensitivity. Due to their unique advantages of excellent catalytic properties and high affinity for heavy metal ions, functional nucleic acids such as DNAzymes and aptamers show great promise in the development of novel sensors for heavy metal ion assays. In this review, we summarize the development of functional nucleic acid-based sensors for the detection of Pb(2+), Hg(2+) and Ag(+), and especially focus on two categories including the direct assay and the amplification-based assay. We highlight the emerging trends in the development of sensitive and selective sensors for heavy metal ion assays as well.

Homogeneous bioluminescence detection of biomolecules using target-triggered hybridization chain reaction-mediated ligation without luciferase label.

We develop a new homogeneous method for sensitive detection of various biomolecules on the basis of bioluminescence monitoring the released AMP from the target-triggered hybridization chain reaction-mediated ligation. The introduction of hybridization chain reaction not only improves the sensitivity of DNA assay, but also facilitates the sensitive detection of proteins by designing specific aptamer triggers, providing a universally amplified platform for simultaneous detection of different kinds of biomolecules. Importantly, this bioluminescence assay employs the target-dependent ATP from the ligation byproduct of AMP as the reporter without the requirement for the sophisticated luciferase manipulation, complicated immobilization, and separation steps. The proposed method has significant advantages of simplicity, high sensitivity, low cost, and high throughput, and holds a great promise for practical point-of-care applications.

A novel SERS-based magnetic aptasensor for prostate specific antigen assay with high sensitivity.

The accurate and highly sensitive detection of prostate specific antigen (PSA) is particularly important, especially for obese men and patients. In this report, we present a novel aptamer-based surface-enhanced Raman scattering (SERS) sensor that employs magnetic nanoparticles (MNPs) core-Au nanoparticles (AuNPs) satellite assemblies to detect PSA. The high specific biorecognition between aptamer and PSA caused the dissolution of the core-satellite assemblies, thus the concentration of functionalized AuNPs (signal probes) existing in the supernatant was on the rise with the continual addition of PSA. The aptamer-modified MNPs were used as supporting materials and separation tools in the present sensor. With the assistance of magnet, the mixture was removed from the supernatant for the concentration effects. It was found that the corresponding SERS signals from the supernatant were in direct correlation to PSA concentrations over a wide range and the limit of detection (LOD) was as low as 5.0pg/mL. Excellent recovery was also obtained to assess the feasibility of this method for human serum samples detection. All of these results show a promising application of this method. And this novel sensor can be used for the accurate and highly sensitive detection of PSA in clinic samples in the future.

A single quantum dot-based biosensor for telomerase assay.

Telomerase is a promising biomarker and a therapeutic target due to its extensive expression in human tumors such as lung cancer and breast cancer. Here, we develop a single quantum dot (QD)-based biosensor for the sensitive detection of telomerase activity. This single QD-based biosensor has significant advantages of simplicity and high sensitivity, and it can be applied for the discrimination of tumor cells from normal cells as well as the screening of anticancer drugs.

Sensitive detection of point mutation using exponential strand displacement amplification-based surface enhanced Raman spectroscopy.

Accurate identification of point mutation is particularly imperative in the field of biomedical research and clinical diagnosis. Here, we develop a sensitive and specific method for point mutation assay using exponential strand displacement amplification (SDA)-based surface enhanced Raman spectroscopy (SERS). In this method, a discriminating probe and a hairpin probe are designed to specifically recognize the sequence of human K-ras gene. In the presence of K-ras mutant target (C→T), the 3'-terminal of discriminating probe and the 5'-terminal of hairpin probe can be ligated to form a SDA template. Subsequently, the 3'-terminal of hairpin probe can function as a primer to initiate the SDA reaction, producing a large amount of triggers. The resultant triggers can further hybridize with the discriminating probes to initiate new rounds of SDA reaction, leading to an exponential amplification reaction. With the addition of capture probe-modified gold nanoparticles (AuNPs) and the Rox-labeled reporter probes, the amplified triggers can be assembled on the surface of AuNPs through the formation of sandwich hybrids of capture probe-trigger-reporter probe, generating a strong Raman signal. While in the presence of K-ras wild-type target (C), neither ligation nor SDA reaction can be initiated and no Raman signal is observed. The proposed method exhibits high sensitivity with a detection limit of 1.4pM and can accurately discriminate as low as 1% variant frequency from the mixture of mutant target and wild-type target. Importantly, this method can be further applied to analyze the mutant target in the spiked HEK293T cell lysate, holding great potential for genetic analysis and disease prognosis.

Simultaneous detection of mercury(II) and silver(I) ions with picomolar sensitivity.

We develop a new method for the simultaneous detection of Hg(2+) and Ag(+) by integrating bifunctional strand displacement amplification and multiplexed short linear quencher-fluorophore probes. This method exhibits excellent specificity and high picomolar sensitivity, which is improved by up to 4 orders of magnitude compared to the current approaches for metal ion assay.

Sensitive detection of polynucleotide kinase using rolling circle amplification-induced chemiluminescence.

We develop a new method for the sensitive detection of polynucleotide kinase (PNK) using rolling circle amplification-induced chemiluminescence. This method exhibits high sensitivity with a detection limit of 2.20 × 10(-4) U mL(-1), which is superior to most reported approaches. Moreover, this method can be used to screen both the inhibitors and the activators of PNK, and can be further applied for real sample analysis.

Highly sensitive detection of zearalenone in feed samples using competitive surface-enhanced Raman scattering immunoassay.

Accurate and quantitative analysis of mycotoxin (such as zearalenone) is particularly imperative in the field of food safety and animal husbandry. Here, we develop a sensitive and specific method for zearalenone detection using competitive surface-enhanced Raman scattering (SERS) immunoassay. In this assay, a functional gold nanoparticle was labeled with the Raman reporter and the zearalenone antibody, and a modified substrate was assembled with the zearalenone-bovine serum albumin. With the addition of free zearalenone, the competitive immune reaction between free zearalenone and zearalenone-bovine serum albumin was initiated for binding with zearalenone antibody labeled on gold nanoparticle, resulting in the change of SERS signal intensity. The proposed method exhibits high sensitivity with a detection limit of 1 pg/mL and a wide dynamic range from 1 to 1000 pg/mL. Furthermore, this method can be further applied to analyze the multiple natural feed samples contaminated with zearalenone, holding great potential for real sample detection.

A quantum dot-based microRNA nanosensor for point mutation assays.

We have developed a quantum dot-based microRNA nanosensor for point mutation assays using primer generation-mediated rolling circle amplification. The proposed method exhibits high sensitivity with a detection limit of as low as 50.9 aM and a large dynamic range of 7 orders of magnitude from 0.1 fM to 1 nM. Importantly, this method can be further applied to analyze the point mutation of mir-196a2 in the lung tissues of non small-cell lung cancer patients.

Sensitive detection of methylated DNA using the short linear quencher-fluorophore probe and two-stage isothermal amplification assay.

DNA methylation is an important epigenetic modification of genomes and is associated with various human diseases. Here, we develop a sensitive approach for DNA methylation assay using the short linear quencher-fluorophore DNA probe (QF probe) and two-stage isothermal amplification. With bisulfite treatment, the methylated DNA target is able to hybridize with the template to initiate the strand displacement amplification (SDA), generating abundant triggers which can further hybridize with the QF probes to form the DNA duplexes. The subsequent recognition of DNA duplexes and the cleavage of QF probes by the nicking enzyme can initiate the nicking enzyme signal amplification (NESA), inducing a significant fluorescence enhancement. While in the absence of methylated DNA, neither SDA nor NESA reaction can be initiated and no fluorescence enhancement is observed. This method exhibits high sensitivity with a detection limit of 0.78 pM, and can distinguish as low as 0.1% methylation level from the mixture of methylated and unmethylated DNA. Notably, the introduction of SDA into NESA can improve the detection sensitivity by up to 2 orders of magnitude as compared with the NESA assay, and it can even discriminate single-base mismatched methylated DNA.

Highly sensitive detection of clenbuterol using competitive surface-enhanced Raman scattering immunoassay.

In this report, we present a novel approach to detect clenbuterol based on competitive surface-enhanced Raman scattering (SERS) immunoassay. Herein, a SERS nanoprobe that relies on gold nanoparticle (GNP) is labeled by 4,4'-dipyridyl (DP) and clenbuterol antibody, respectively. The detection of clenbuterol is carried out by competitive binding between free clenbuterol and clenbuterol-BSA fastened on the substrate with their antibody labeled on SERS nanoprobes. The present method allows us to detect clenbuterol over a much wider concentration range (0.1-100 pg mL(-1)) with a lower limit of detection (ca. 0.1 pg mL(-1)) than the conventional methods. Furthermore, by the use of this new competitive SERS immunoassay, the clenbuterol-BSA (antigen) is chosen to fasten on the substrate instead of the clenbuterol antibody, which could reduce the cost of the assay. Results demonstrate that the proposed method has the wide potential applications in food safety and agonist control.