DNA-templated Copper Nanoparticles Research Articles

Electrochemical aptasensor based on DNA-templated copper nanoparticles and RecJf exonuclease-assisted target recycling for lipopolysaccharide detection.

An electrochemical aptasensor for detecting lipopolysaccharides (LPS) was fabricated based on DNA-templated copper nanoparticles (DNA-CuNPs) and RecJf exonuclease-assisted target recycling. The DNA-CuNPs were synthesized on a double-stranded DNA template generated through the hybridization of the LPS aptamer and its complementary chain (cDNA). In the absence of LPS, the CuNPs were synthesized on DNA double-strands, and a strong readout corresponding to the CuNPs was achieved at 0.10 V (vs. SCE). In the presence of LPS, the fabricated aptamer could detach from the DNA double-strand to form a complex with LPS, disrupting the template for the synthesis of CuNPs on the electrode. Meanwhile, RecJf exonuclease could hydrolyze the cDNA together with this single-stranded aptamer, releasing the LPS for the next round of aptamer binding, thereby enabling target recycling amplification. As a result, the electrochemical signal decreased and could be used to indicate the LPS content. The fabricated electrochemical aptasensor exhibited an extensive dynamic working range of 0.01 pg mL-1 to 100 ng mL-1, and its detection limit was 6.8 fg mL-1. The aptasensor also exhibited high selectivity and excellent reproducibility. Moreover, the proposed aptasensor could be used in practical applications for the detection of LPS in human serum samples.

Highly sensitive detection of telomerase based on poly-adenine primer and DNA-templated copper nanoparticles

Telomerase is a potential diagnostic biomarker for a variety of cancers. Development of sensitive and convenient methods to measure the activity of telomerase is of great importance. Herein, an electrochemical method is fabricated with self-assembled poly-adenine (polyA) based primer. Target telomerase catalyzes the extension of primer and the product with multiple repeated sequences captures abundant DNA-templated copper nanoparticles (CuNPs). The nanomaterials act as electrochemical signal sources. CuNPs can be further dissolved and detected with significant voltammetric responses. This developed electrochemical biosensor exhibits a wide dynamic range from 10 to 5 × 104 cell/mL and low limit of detection (2 cells/mL). The satisfactory performances are due to the excellent electrochemical properties of CuNPs and target induced enrichment effect. This method provides an effective approach for the telomerase activity monitoring in cancer diagnostics.

Target Enzyme-Triggered Click Chemistry and Hybridization Chain Reaction for Fluorescence Nonculture Homogeneous Analysis of E. coli in Bloodstream Infections.

Escherichia coli is the major pathogen that causes bloodstream infections (BSI). It is critical to develop nonculture identification methods which can meet the urgent need of clinical diagnosis and treatment. In this study, we reported a homogeneous fluorescence E. coli analysis system using β-galactosidase (β-Gal) as the biomarker and double-stranded DNA-templated copper nanoparticles (dsDNA-Cu NPs) as the signal output. The product of the enzymatic hydrolysis reaction, p-aminophenol (PAP), could reduce Cu2+ to Cu+, triggering the alkyne-azido cycloaddition reaction (CuAAC). Subsequently, the hybrid chain reaction (HCR) was initiated, producing the dsDNA template used to generate Cu NPs in situ. The system achieved a wide linear range for β-Gal and E. coli 1-104 mU/L and 10-2-10 colony-forming unit (CFU)/mL, and a detection limit of 0.3 mU/L and 0.003 CFU/mL, respectively. 65 samples (45 blood and 20 urine) were collected to evaluate the clinical practicality. The results demonstrated remarkable area under the curve (AUC) values of 0.95 and 0.916 from uncultured urine and blood, respectively. It had 100% specificity and 83.3% sensitivity. The whole duration of the strategy was 3.5 h, which significantly reduced the turnaround time (TAT) and facilitated early BSI diagnosis to improve patients' prognosis. Our work had the potential to be an alternative to culture-based methods in clinics.

A Fluorescent Biosensor for Streptavidin Detection Based on Double-Hairpin DNA-Templated Copper Nanoparticles

In this paper, we developed a sensitive, label-free and facile fluorescent strategy for detecting streptavidin (SA) based on double-hairpin DNA-templated copper nanoparticles (CuNPs) and terminal protection of small molecule-linked DNA. Herein, a special DNA hairpin probe was designed and synthesized, which contained two poly T single-stranded loops and a nick point in the middle of the stem. Inspired by the concept of the terminal protection interaction, the specific binding of SA to the biotinylated DNA probe can prevent the exonuclease degradation and keep the integrity of DNA probe, which can be used for synthesizing fluorescent CuNPs as a template. Conversely, the DNA probe would be digested by exonucleases and therefore, would fail to form CuNPs without SA. After systematic optimization, the detection range of SA concentration is from 0.5 to 150 nM with a low detection limit of 0.09 nM. Additionally, the proposed method was also successfully applied in the biological samples. Finally, the proposed method is sensitive, effective and simple, and can be potentially applied for predicting diseases and discovering new drugs.

Ratiometric fluorescent detection of pesticide based on split aptamer and magnetic separation

Pesticide residues have seriously threatened human health and affected the sustainable development of the environment. It is pivotal to establish an efficient detection platform for pesticide assays. A novel ratiometric fluorescent aptasensor for sensitive determination of pesticide has been successfully established. Carbon dots (CDs) are used as the reference emitter, while the T-rich single-stranded DNA templated copper nanoparticles (CuNPs) provide the increasing signal. More precisely, CDs/magnetic beads (MBs) combined with split aptamer are used in this detection platform to present a label-free and enzyme-free isocarbophos (ICP) detection with high sensitivity and specificity. In the presence of ICP, a sandwich-typed complex is formed which attaches a T-rich DNA. After magnetic separation and addition of Cu2+/ascorbic acid (AA), the CuNPs are generated and the strong red luminescent appears. Without ICP, just blue emission from CDs exits. The developed ratiometric aptasensor offers an excellent ICP detection in the linear range of 3–200 nM with a detection limit of 0.81 nM. It provides new ways for the assay of pesticides in agricultural products or environmental samples.

Fluorescence determination of glyphosate based on a DNA-templated copper nanoparticle biosensor.

Arapid and convenient fluorescence glyphosate (GLYP) biosensor was developed based on DNA-templated copper nanoparticles (DNA-CuNPs). In the absence of GLYP, the DNA-CuNPs were formed through the reduction of Cu2+ by vitamin C (Vc). TheDNA-CuNPs emitted intense fluorescence at 615nm when being excited at 340nm. In the presence of GLYP, GLYP can strongly chelate with Cu2+ by the phosphate and carboxyl groups to decrease the amount of free Cu2+. Due to the lack of free Cu2+, DNA-CuNPs cannot be formed, which caused the fluorescence to decrease. The whole detection process of this proposed GLYP biosensor can becompleted within 14min. Titration experiments showed that this biosensor had a linear relationship for GLYP in therange 1 to 18µM with a limit of detection (LOD) of 0.47µM. This biosensor showed obvious selectivity among other pesticides, even between GLYP and organophosphorus pesticides. This biosensor performed well for GLYP detection in real samples with recoveries of 88.0-104.0%.

A versatile fluorescent nanobeacon lighted by DNA-templated copper nanoparticles and the application in isothermal amplification detection

In this work, an environmentally-friendly and versatile nanobeacon was constructed by utilizing DNA-templated copper nanoparticles (CuNPs) as fluorescence signal source. As the key component of the nanobeacon, a hairpin DNA was designed to contain four segments: two segments for CuNPs template sequence, a target recognition segment and a blocking segment. At room temperature, the target recognition segment partly hybridizes with the blocking segment and thus prohibits the formation of double stranded DNA template, so that no CuNPs can be generated on the hairpin DNA. While a target is introduced, the specific binding of target with recognition sequence triggers off the conformational transformation of the hairpin DNA, which contributes to the formation of the CuNPs template. As a result, the in-situ generation of CuNPs gives birth to the fluorescence signal readout that can be used to identify the target. By reasonably varying the recognition sequence within hairpin DNA, a series of nanobeacons in response to corresponding targets, such as DNA, microRNA, thrombin, and ATP, were put forward with satisfactory sensitivity and selectivity. Moreover, this nanobeacon was also integrated with the strategy of enzyme-assisted target-recycling to realize signal amplification and ultrasensitive detection, which further demonstrated the versatility of the nanobeacon. This novel nanobeacon is expected to be a promising alternative to classical dye-labeled molecular beacon and provide new perspective on ultrasensitive fluorescence sensing.

Fluorescence resonance energy transfer using DNA-templated copper nanoparticles for ratiometric detection of microRNAs.

We examined the effectiveness of a ratiometric method using DNA-templated copper nanoparticles, which can function as a probe for fluorescence resonance energy transfer. This method in combination with PCR successfully detected the target microRNA, which corresponded well with the results obtained by quantitative reverse transcription PCR.

Highly specific nuclear labeling via in situ formation of fluorescent copper nanoparticles.

When imaging cells, nuclear counterstaining is imperative; however, many commercial nuclear-staining dyes based on nucleic acid intercalation result in nonspecific signals in the cytoplasm. Here, we propose a new strategy that stains the nucleus with high specificity by in situ formation of DNA-templated copper nanoparticles (CuNPs). We demonstrated that genomic DNA in the nucleus enabled rapid formation of highly fluorescent CuNPs immediately following addition of a copper ion source and ascorbate as a reducing agent. Moreover, we found that RNA and mitochondrial DNA, largely responsible for nonspecific cytoplasmic signals from commercial nuclear-staining dyes, did not mediate the formation of the highly fluorescent CuNPs, resulting in highly specific nuclear staining at a reduced cost relative to commercially available methods. Furthermore, we verified the compatibility of the proposed method with other fluorescence-labeling techniques. These results demonstrated the efficacy of this method and its promise as a powerful tool for cell imaging.

A fluorescent aptasensor for sensitive detection of isocarbophos based on AT-rich three-way junctions DNA templated copper nanoparticles and Fe3O4@GO

A novel fluorescent aptasensor for sensitive determination of pesticide was successfully established, using AT-rich three-way junctions (AT-TWJ) DNA stabilized copper nanoparticle (CuNP) as a fluorescent signal and magnetic Fe3O4@GO as a single-stranded DNA (ssDNA) adsorbent to reduce the detection background. The detection just involves one strand of dsDNA and two strands of ssDNA. The dsDNA is hybridized by the target aptamer and the partially complementary DNA, acting as a recognition unit. After the specific combination between targets with its aptamer, the released ssDNA and the other two ssDNA can assemble into AT-TWJ DNA. The added Fe3O4@GO cannot adsorb the nascent blunt-ended AT-TWJ DNA. Then, the inputting of ascorbic acid (AA) and Cu2+ into the supernatant facilitates the formation of AT-TWJ DNA templated CuNPs, generating strong fluorescence. The developed strategy presents a sensitive and specific isocarbophos detection in the linear range of 10–500 nM with a detection limit of 3.38 nM, providing a new path for the detection of pesticides in the fields of food and environmental monitoring.

Handheld fluorometer for in-situ melamine detection via interference synthesis of dsDNA-templated copper nanoparticles.

Fluorescent copper nanoparticles templated by dsDNA have gained significant research interest as they are inexpensive and easy to synthesize, and have found applications in the detection of a wide range of analytes. The presence of the analyte in the reaction mixture interferes with the synthesis of the copper nanoparticles and the subsequent drop in fluorescence can be correlated to the concentration of the analyte present in the solution. Analyte detection using copper nanoparticle-based assays is amenable for in-situ applications as the test does not require expensive reagents and can be performed at room temperature. However, expensive and sophisticated detection systems are required for the detection of copper nanoparticles due to the low fluorescence emission signal from these nanoparticles. This restricts the use of the technology to centralized labs. Utilizing a recently developed chemical technique for fluorescence enhancement, this paper presents the first report of a handheld fluorometer capable of detecting DNA-templated copper nanoparticles. The fluorometer is portable and constructed with low-cost, off-the-shelf components like a UV-LED and a PIN photodiode. The performance of the developed system is demonstrated through the detection of melamine in milk samples via the interference synthesis of copper nanoparticles. Melamine is an adulterant used in dairy products that is harmful to human health if present in levels above 1ppm. The developed system is capable of detecting up to 0.1ppm of melamine in milk samples with a linear relationship observed between the detector output and concentration of melamine in the range from 0.1ppm to 100ppm (R2=0.9979).

Detection of copper nanoparticles templated by DNA using etched Fibre Bragg Grating sensor

In recent years, DNA-templated copper nanoparticles have been used to detect several important analytes, primarily through fluorescence spectrometry. However, the fluorescence of these nanoparticles decays within a few minutes making the quantitative estimation of analytes difficult. Despite the loss of fluorescence, the nanoparticles remain intact as evidenced by microscopy techniques like TEM and AFM. Here, etched fibre Bragg Grating (eFBG) sensors are presented as a detection/characterization tool for copper nanoparticles templated by double stranded DNA. The proposed sensor has a sensitivity of 1.5395 nm/RIU and a resolution of 0.00612 RIU. The formation of the copper nanoparticles on the DNA template attached to the eFBG sensor leads to a significant shift of ≈ 58 pm in the reflected Bragg wavelength signal. Further, the application of the proposed eFBG sensors for analyte detection utilizing the interruption in the synthesis of copper nanoparticles is demonstrated here with the example of melamine detection. The Bragg wavelength shift varies linearly with the log concentration of melamine ( ${R}^{2}=0.9482$ ) and the sensor can detect up to 0.1 ppm of melamine.

DNA-templated copper nanoparticles as signalling probe for electrochemical determination of microRNA-222.

An ultrasensitive electrochemical biosensor is described for the determination of microRNAs. It is based on the use of DNA-templated copper nanoparticles (Cu NPs) as signalling probe. MicroRNA-222 was selected as the model analyte. The probe was obtained from two different oligonucleotides (containing complementary bases) via hybridization chain reaction to form long DNA concatemers as template. The Cu NPs were formed by reaction of ascorbate with copper sulfate. The biosensor was fabricated as follows: (a) Capture probe (cDNA) with a thiolated group was immobilized on reduced graphene oxide modified with gold nanoparticles (rGO/Au NPs), (b) materials was placed on a glassy carbon electrode (GCE); (c) the modified electrode (cDNA/rGO/Au NPs/GCE) was sequentially hybridized with microRNA-222 and signal probe; this results in the formation of a sandwich structure of cDNA-microRNA-signal probe on surface of the modified electrode. Differential pulse voltammetry was employed to record the electrochemical response of biosensor in pH6.0 solution. As a result, a sensitive oxidation current with a peak potential at 0.10V (vs. SCE) was obtained corresponding to Cu NPs. The experimental conditions were optimized. Under optimal conditions, the biosensor exhibited wide linear response range (0.5 fM to 70nM) and low limit of detection (0.03 fM; at S/N = 3). The assay possesses high selectivity and can discriminate analyte microRNA from single-base mismatched microRNA. Graphical abstractA sensitive electrochemical biosensor is describedfor the determination ofmicroRNA-222 by using a dsDNA-templated Cu NPs as signalling probe. (A) represents the preparation of signal probe, and (B) represents the fabrication of electrochemical microRNA sensor.

Target-induced in-situ formation of fluorescent DNA-templated copper nanoparticles by a catalytic hairpin assembly: application to the determination of DNA and thrombin.

A fluorometric method is described for nucleic acid signal amplification through target-induced catalytic hairpin assembly with DNA-templated copper nanoparticles (Cu NPs). The toehold-mediated self-assembly of three metastable hairpins is triggered in presence of target DNA. This leads to the formation of a three-way junction structure with protruding mononucleotides at the 3' terminus. The target DNA is released from the formed branched structure and triggers another assembly cycle. As a result, plenty of branched DNA becomes available for the synthesis of Cu NPs which have fluorescence excitation/emission maxima at 340/590nm. At the same time, the branched structure protects the Cu NPs from digestion by exonuclease III. The unreacted hairpins are digested by exonuclease III, and this warrants a lower background signal. The method can detect ssDNA (24nt) at low concentration (44 pM) and is selective over single-nucleotide polymorphism. On addition of an aptamer, the strategy can also beapplied to the quantitation of thrombin at levels as low as 0.9nM. Graphical abstractSchematic representation of target-induced catalytic hairpin assembly to form branched DNA template for the in situ synthesis of fluorescent Cu nanoparticles.

Synthesis of DNA-templated copper nanoparticles with enhanced fluorescence stability for cellular imaging.

Fluorescence of DNA-templated copper nanoparticles (DNA-CuNPs) is not stable over time which limits applications in cellular imaging. This is due to the presence of oxygen during synthesis which oxidizes Cu(0) to Cu(II) and also produces the free hydroxyl radical. The authors have prepared DNA-CuNPs with enhanced temporal stability of fluorescence by optimizing the reaction conditions so as to minimize the deleterious effects of oxygen. The operational lifetime of DNA-CuNPs was increased from 25min to 200min. Fluorescence spectra of DNA-CuNPs in optimized condition show an emission peak at 650nm when excited at 340nm. DNA-CuNPs synthesized in this manner were used for cell imaging. As a proof of concept, the nucleus of a human colon cell line (HCT116) was stained. The method does not involve any chemicals other that copper sulfate and ascorbate. This new approach for generating DNA-CuNPs improves imaging of biological processes and provides a basis for developing other types of DNA-templated nanomaterials. Graphical abstract Schematic presentation of the formation of fluorescent DNA-templated copper nanoparticles (DNA-CuNPs). A large amount of ascorbate provides long operational lifetime for cellular imaging under the condition exposed to oxygen. *Asc- and **DHA stand for ascorbate and dehydroascorbic acid.

A label-free and enzyme-free fluorescent aptasensor for sensitive detection of acetamiprid based on AT-rich dsDNA-templated copper nanoparticles

A label-free and enzyme-free aptasensor for sensitive assay of acetamiprid has been established using AT-rich double-stranded (ds) DNA-templated copper nanoparticles (CuNPs) as fluorescent probe. In this work, two hairpin DNA, HP1 and HP2, were elaborately designed with AT-rich DNA sequences in their loops. The aptamer of acetamiprid was located at the 3′-terminal of HP1, which was caged in the stem of HP1. Upon the addition of acetamiprid, the aptamer could combine with acetamiprid to form a target/aptamer complex, and thus its free 5′-terminal was released. Subsequently, the protruded 3′-terminal of HP2 could hybridize with the free 5′-terminal of HP1 to form a stable AT-rich dsDNA. When it interacted with Cu2+ and ascorbic acid (AA), the AT-rich dsDNA/CuNPs were generated with strong fluorescence, offering a “switch-on” detection of acetamiprid. The developed strategy could high selectively detect acetamiprid at the concentration as low as 2.37 nM. Moreover, the possibility of this strategy for the food sample analysis was also investigated. The obtained results demonstrate that the developed strategy has a promising application potential for acetamiprid assay in food safety fields.

Enhancement of the fluorescence properties of double stranded DNA templated copper nanoparticles

The weak fluorescence emission from dsDNA templated copper nanoparticles necessitates the use of high-end detectors like photomultiplier tubes for their detection. This sets limitations on their applicability to in-situ analyte detection and point-of-care applications which utilize comparatively low cost and less sensitive detectors. In this article, a technique to improve the fluorescence properties of copper nanoparticles templated on dsDNA is reported. The fluorescence enhancement is achieved by introducing a modification in the conventional synthesis technique by using a combination of sodium ascorbate and Taq buffer. When compared to the existing methods, the proposed method achieves 11 times higher fluorescence signal intensity from the dsDNA templated copper nanoparticles and 4 times faster attainment of maximum fluorescence signal. The effect of the ionic strength of the individual constituent components of Taq buffer on the fluorescence emission from the copper nanoparticles is also studied here. The utility of this enhancement strategy for analyte measurement is demonstrated with the example of melamine detection from milk samples. A linear relationship was observed between the fluorescence intensity from the copper nanoparticles and the concentration of melamine in the range from 0.5 ppm to 100 ppm (R2 = 0.9919), with a limit of detection of 0.1 ppm. The reported fluorescence enhancement technique also results in 2.95 times improved sensitivity of detection when compared to the conventional technique.

In situ formation of DNA-templated copper nanoparticles as fluorescent indicator for hydroxylamine detection.

Herein, we develop a facile method for selective and sensitive detection of hydroxylamine (HA) based on the in situ formation of DNA templated copper nanoparticles (DNA-CuNPs) as fluorescent probes. It is firstly found that HA as a reducing agent can play a key role in the in situ formation of fluorescent DNA-CuNPs. This special optical property of DNA-CuNPs with (λex = 340 nm, λem = 588 nm) with a mega-Stokes shifting (248 nm) makes it applicable for the turn-on detection of HA. In addition, this fluorescent method has several advantages such as being simple, rapid, and environmentally friendly, because it avoids the traditional organic dye molecules and complex procedures. Under optimized conditions, this platform achieves a fluorescent response for HA with a detection limit of 0.022 mM. Especially, successful detection capability in tap waters and ground waters exhibits its potential to be general method.

Substrate specificity-enabled terminal protection for direct quantification of circulating MicroRNA in patient serums.

Currently, reported affinity pairings still lack in diversity, and thus terminal protection relying on steric hindrance is restricted in designing nucleic acid-based analytical systems. In this work, resistance to exonuclease is testified by group modification or backbone replacement, and the 3'-phosphate group (P) reveals the strongest exonuclease I-resistant capability. Due to the substrate specificity of enzymatic catalysis, this 3'-P protection works in a "direct mode". By introducing DNA templated copper nanoparticles, an alkaline phosphatase assay is performed to confirm the 3'-P protection. To display the application of this novel terminal protection, a multifunctional DNA is designed to quantify the model circulating microRNA (hsa-miR-21-5p) in serums from different cancer patients. According to our data, hsa-miR-21-5p-correlated cancers can be evidently distinguished from non-correlated cancers. Meanwhile, the effect of chemotherapy and radiotherapy on breast cancer is evaluated from the perspective of hsa-miR-21-5p residue in serums. Since greatly reducing the limitations of DNA design, this P-induced terminal protection can be facilely integrated with other DNA manipulations, thereby constructing more advanced biosensors with improved analytical performances for clinical applications.

Double G-quadruplexes in a copper nanoparticle based fluorescent probe for determination of HIV genes.

A DNA-templated copper nanoparticle (CuNP) probe has been developed for the determination of the human immunodeficiency virus oligonucleotide (HIV-DNA). The function of the probe relies on affinity binding-induced DNA hybridization associated with the use of double G-quadruplexes. Double-stranded DNA(dsDNA) with poly(AT-TA) bases was used as a template for synthesis of dsDNA-CuNPs. These have weak fluorescence. In the next step, two G-rich sequences that are linked to both sides of the ds-DNA are locked by HIV complementary DNA (cDNA). If HIV-DNA is introduced, it will hybridize with cDNA, thereby transforming the two G-rich sequences into G-quadruplexes. This enhances the fluorescence of the adjacent dsDNA-CuNPs. Fluorescence increases linearly in the 1 to 200 and 250-1000nM HIV-DNA concentration range, and the detection limit is 13 pM. This enzyme-free fluorometric assay is time-saving, easily operated, and therefore has large potential in biosensing because it may be extended to various other DNA targets. Graphic abstract Double-strand DNA-templated copper nanoparticles (DNA-CuNPs) have weak fluorescence. When Human Immunodeficiency Virus oligonucleotide (HIV-DNA) is added, it completely hybridized with HIV complementary DNA (cDNA). As a result, the two exposed G-rich sequences aretransformed into G-quadruplexes, and an apparent increase in the fluorescence intensity can be observed. (AA: ascorbic acid).