Formation Of Double-stranded DNA Research Articles

A Novel PCR-Free Ultrasensitive GQD-Based Label-Free Electrochemical DNA Sensor for Sensitive and Rapid Detection of Francisella tularensis.

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two-five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor's detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Comparison of Double-Stranded DNA at the 5' and 3' Ends of the G-Triplex and Its Application in the Detection of Hg(II).

Leveraging the fluorescence enhancement effect of the G-triplex (G3)/thioflavin T (ThT) catalyzed by the adjacent double-stranded DNA positioned at the 5' terminus of the G3, the G3-specific oligonucleotide (G3MB6) was utilized to facilitate the rapid detection of mercury (Hg(II)) through thymine-Hg(II)-thymine (T-Hg(II)-T) interactions. G3MB6 adopted a hairpin structure in which partially complementary strands could be disrupted with the presence of Hg(II). It prompted the formation of double-stranded DNA by T-Hg(II)-T, inducing the unbound single strand of G3MB6 to spontaneously form a parallel G3 structure, producing a solid fluorescence signal by ThT. Conversely, fluorescence was absent without Hg(II), since no double strand and formation of G3 occurred. The fluorescence intensity of G3MB6 exhibited a positive correlation with Hg(II) concentrations from 17.72 to 300 nM (R2 = 0.9954), boasting a notably low quality of limitation (LOQ) of 17.72 nM. Additionally, it demonstrated remarkable selectivity for detecting Hg(II). Upon application to detect Hg(II) in milk samples, the recovery rates went from 100.3% to 103.2%.

Dual-mode fluorescence and electrochemiluminescence sensors based on Ru-MOF nanosheets for sensitive detection of apoE genes.

A fluorescence-electrochemiluminescence (FL-ECL) dual-mode sensor for apoE gene detection has been developed, leveraging the unique properties of ruthenium metal organic framework nanosheets (RuMOFNSs). The system utilizes the quenching effect of the Ru(bpy)32+ ECL signal by ferrocene, leading to the synthesis of a multi-electron electrical signal marker, bisferrocene. By immobilizing the P-DNA on RuMOFNSs, bisferrocene quenches both FL and ECL signals. The addition of T-DNA and the consequent formation of double-stranded DNA enable the ExoIII enzyme to excise the bisferrocene fragment, restoring the signals. The sensor demonstrates wide detection linear ranges (1 fM to 1 nM for FL and 0.01 fM to 10 pM for ECL) and remarkable sensitivity (0.048 fM for FL and 0.016 fM for ECL). The dual-mode design offers enhanced reliability through a self-correction feature, reducing false positives. Compared to single-mode sensors, the dual-mode sensor shows significant advantages. Real-world testing confirms the sensor's capacity for robust detection in actual samples, underscoring its promising application in early disease diagnosis. This innovative approach opens up avenues for multi-signal response sensors, offering significant potential for diagnostic technologies.

ILF3 safeguards telomeres from aberrant homologous recombination as a telomeric R-loop reader.

Telomeres are specialized structures at the ends of linear chromosomes that protect genome stability. The telomeric repeat-containing RNA (TERRA) that is transcribed from subtelomeric regions can invade into double-stranded DNA regions and form RNA:DNA hybrid-containing structure called R-loop. In tumor cells, R-loop formation is closely linked to gene expression and the alternative lengthening of telomeres (ALT) pathway. Dysregulated R-loops can cause stalled replication forks and telomere instability. However, how R-loops are recognized and regulated, particularly at telomeres, is not well understood. We discovered that ILF3 selectively associates with telomeric R-loops and safeguards telomeres from abnormal homologous recombination. Knocking out ILF3 results in excessive R-loops at telomeres and triggers telomeric DNA damage responses. In addition, ILF3 deficiency disrupts telomere homeostasis and causes abnormalities in the ALT pathway. Using the proximity-dependent biotin identification (BioID) technology, we mapped the ILF3 interactome and discovered that ILF3 could interact with several DNA/RNA helicases, including DHX9. Importantly, ILF3 may aid in the resolution of telomeric R-loops through its interaction with DHX9. Our findings suggest that ILF3 may function as a reader of telomeric R-loops, helping to prevent abnormal homologous recombination and maintain telomere homeostasis.

Prevalence and Genomic Sequence Analysis of Domestic Cat Hepadnavirus in the United States.

Hepadnaviruses are partially double-stranded DNA viruses that infect a variety of species. The prototypical virus in this family is the human hepatitis B virus, which chronically infects approximately 400 million people worldwide and is a risk factor for progressive liver disease and liver cancer. The first hepadnavirus isolated from carnivores was a domestic cat hepadnavirus (DCH), initially identified in Australia and subsequently detected in cats in Europe and Asia. As with all characterized hepadnaviruses so far, DCH infection has been associated with hepatic disease in its host. Prevalence of this infection in the United States has not been explored broadly. Thus, we utilized conventional and quantitative PCR to screen several populations of domestic cats to estimate DCH prevalence in the United States. We detected DCH DNA in 1 out of 496 animals (0.2%) in the U.S. cohort. In contrast, we detected circulating DCH DNA in 7 positive animals from a cohort of 67 domestic cats from Australia (10.4%), consistent with previous studies. The complete consensus genome of the U.S. DCH isolate was sequenced by Sanger sequencing with overlapping PCR products. An in-frame deletion of 157 bp was identified in the N-terminus of the core open reading frame. The deletion begins at the direct repeat 1 sequence (i.e., the 5′ end of the expected double-stranded linear DNA form), consistent with covalently closed circular DNA resultant from illegitimate recombination described in other hepadnaviruses. Comparative genome sequence analysis indicated that the closest described relatives of the U.S. DCH isolate are those previously isolated in Italy. Motif analysis supports DCH using NTCP as an entry receptor, similar to human HBV. Our work indicates that chronic DCH prevalence in the U.S. is likely low compared to other countries.

A new method for determination of polysaccharides in adsorption of Hg2+

Bacterial extracellular polysaccharides (EPS) has a good heavy metal ion adsorption function and can be used in sewage treatment of heavy metal pollution. In this work, we develop a new, label-free, turn-on fluorescent sensor for detecting EPS adsorbed Hg2+. This approach is based on the conformational switch from G-quadruplex complex induced by fluorogenic dye Thioflavin T (ThT) to Hg2+ mediated T–Hg–T double-stranded DNA formation. By this method, Hg2+ in aqueous solution can be detected by fluorescence spectroscopy in the range of 5–1200 nM. The entire reaction and analysis of Hg2+ was performed in 10 min under optimal experimental conditions (1.4 μM ThT, 0.35 μM TBA, 1.2 μM Hg2+ and 10 min incubation time at 25℃, pH7.5). Then, we have utilized this facile approach to detect the adsorption properties of different EPS on Hg2+ with fluorescence spectrometry. These EPS were produced by the fermentation of Pseudoalteromonas sp. BAY, Bacillus sp. RS1, Mesonia sp. Hua40 and Bacillus sp3. SL7 respectively. The Hg2+ adsorption capacity of EPS is between 80.1 mg/g to 169.6 mg/g. Among them, the extracellular polysaccharides of Mesonia sp. Hua40 have the strongest Hg2+ adsorption capacity, about 169.6 mg/g. This method provides an effective detection technique to analyze Hg2+ adsorption capacity of EPS.

Ultrasensitive Label-Free DNA Detection Based on Solution-Gated Graphene Transistors Functionalized with Carbon Quantum Dots.

Developing highly sensitive, reliable, cost-effective label-free DNA biosensors is challenging with traditional fluorescence, electrochemical, and other techniques. Most conventional methods require labeling fluorescence, enzymes, or other complex modification. Herein, we fabricate carbon quantum dot (CQD)-functionalized solution-gated graphene transistors for highly sensitive label-free DNA detection. The CQDs are immobilized on the surface of the gate electrode through mercaptoacetic acid with the thiol group. A single-stranded DNA (ssDNA) probe is immobilized on CQDs by strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, which led to a shift of Dirac voltage and the channel current response. The limit of detection can reach 1 aM which is 2-5 orders of magnitude lower than those of other methods reported previously. The sensor also exhibits a good linear range from 1 aM to 0.1 nM and has good specificity. It can effectively distinguish one-base mismatched target DNA. The response time is about 326 s for the 1 aM target DNA molecules. This work provides good perspectives on the applications in biosensors.

Immobilization-free electrochemical DNA sensor based on signal cascade amplification strategy.

The development of convenient and efficient strategies without using complex nanomaterials or enzymes for signal amplification is very important for bioanalytical applications. Herein, a novel electrochemical DNA sensor was developed by harnessing the signal amplification efficiency of catalytic hairpin assembly (CHA) and a brand-new signal marker tetraferrocene. The prepared sensor had both ends of the probe H2 labeled with tetraferrocene; both ends have a large number of unhybridized T bases, which cause tetraferrocene to move closer to the electrode surface, generating a high-efficiency amplification signal. In the presence of target DNA, it induced strand exchange reactions promoting the formation of double-stranded DNA and recycling of target DNA. Under optimal conditions, the sensor showed a good linear correlation between the peak currents and logarithm of target DNA concentrations (ranging from 0.1 fM to 0.3125 pM) with a detection limit of 0.06 fM, which is obtained by a triple signal-to-noise ratio. Additionally, the prepared sensor possesses excellent selectivity, reproducibility, and stability, demonstrating efficient and stable DNA detection methodology.

A DNA sensor based on upconversion nanoparticles and two-dimensional dichalcogenide materials

We demonstrate the fabrication of a new DNA sensor that is based on the optical interactions occurring between oligonucleotide-coated NaYF4:Yb3+;Er3+ upconversion nanoparticles and the two-dimensional dichalcogenide materials, MoS2 and WS2. Monodisperse upconversion nanoparticles were functionalized with single-stranded DNA endowing the nanoparticles with the ability to interact with the surface of the two-dimensional materials via van der Waals interactions leading to subsequent quenching of the upconversion fluorescence. By contrast, in the presence of a complementary oligonucleotide target and the formation of double-stranded DNA, the upconversion nanoparticles could not interact with MoS2 and WS2, thus retaining their inherent fluorescence properties. Utilizing this sensor we were able to detect target oligonucleotides with high sensitivity and specificity whilst reaching a concentration detection limit as low as 5 mol·L−1, within minutes.

Base-Pairing and Base-Stacking Contributions to Double-Stranded DNA Formation.

Double-stranded (ds)DNA formation and dissociation are of fundamental biological importance. The negative DNA charge influences the dsDNA stability. However, the base pairing and the stacking between neighboring bases are responsible for the sequence-dependent stability of dsDNA. The stability of a dsDNA molecule can be estimated from empirical nearest-neighbor models based on contributions assigned to base-pair steps along the DNA and additional parameters because of DNA termini. In efforts to separate contributions, it has been concluded that base stacking dominates dsDNA stability, whereas base pairing contributes negligibly. Using a different model for dsDNA formation, we reanalyze dsDNA stability contributions and conclude that base stacking contributes already at the level of separate ssDNAs but that pairing contributions drive the dsDNA formation. The theoretical model also predicts that stability contributions of base-pair steps that contain only guanine/cytosine, mixed steps, and steps with only adenine/thymine follow the order 6:5:4, respectively, as expected based on the formed hydrogen bonds. The model is fully consistent with the available stacking data and the nearest-neighbor dsDNA parameters. It allows assigning a narrowly distributed value for the effective free energy contribution per formed hydrogen bond during dsDNA formation of -0.72 kcal·mol-1 based entirely on the experimental data.

Aptamer-Based Colorimetric Probe for trans-Zeatin Detection Using Unmodified Gold Nanoparticle.

Trans-Zeatin is the major active phytohormone in immature corn kernels. Herein, a highly sensitive, good selective and simple aptamer-based colorimetric method for the detection of trans-zeatin was constructed. The selected aptamer sequence binds with trans-zeatin and induces a duplex-to-aptamer structure switching. The gold nanoparticles (AuNPs) solution is stable with high-concentration salt, which is protected by red complementary DNA. In the absence of trans-zeatin, the color of AuNPs changed from red to blue because aptamer DNA and complementary DNA form double-stranded DNA. Thus, the ratio of absorbance intensities (A522/A650) of AuNPs is changed with the concentration of trans-zeatin. The color change could be observed by the naked eye. The linear range of this method covers a large variation of trans-zeatin concentration from 0.05 to 0.75 μM. The detection limit is 0.037 μM. Moreover, this method was applied successfully to detect trans-zeatin in real plant samples.

Dynamic topology of double-stranded telomeric DNA studied by single-molecule manipulation in vitro.

The dynamic topological structure of telomeric DNA is closely related to its biological function; however, no such structural information on full-length telomeric DNA has been reported due to difficulties synthesizing long double-stranded telomeric DNA. Herein, we developed an EM-PCR and TA cloning-based approach to synthesize long-chain double-stranded tandem repeats of telomeric DNA. Using mechanical manipulation assays based on single-molecule atomic force microscopy, we found that mechanical force can trigger the melting of double-stranded telomeric DNA and the formation of higher-order structures (G-quadruplexes or i-motifs). Our results show that only when both the G-strand and C-strand of double-stranded telomeric DNA form higher-order structures (G-quadruplexes or i-motifs) at the same time (e.g. in the presence of 100 mM KCl under pH 4.7), that the higher-order structure(s) can remain after the external force is removed. The presence of monovalent K+, single-wall carbon nanotubes (SWCNTs), acidic conditions, or short G-rich fragments (∼30 nt) can shift the transition from dsDNA to higher-order structures. Our results provide a new way to regulate the topology of telomeric DNA.

Ratiometric fluorescence strategy for p53 gene assay by using nitrogen doped graphene quantum dots and berberine as fluorescence reporters

Herein, a dual-signal ratiometric fluorescence-based p53 gene sensing strategy was developed for the first time. Nitrogen doped graphene quantum dots (NGQDs) were firstly bound with a single-stranded DNA (P1 DNA), which contains berberine aptamer sequence and p53 gene complementary sequence (Cp53 DNA). With the addition of berberine, berberine will bind to berberine aptamer in P1 DNA, and a dual-signal ratiometric fluorescence probe NGQDs/P1 DNA/berberine was established, which displays two fluorescence peaks at 440 nm and 537 nm corresponding to NGQDs and berberine, respectively. P1 DNA can be hydrolyzed by Exonuclease I (Exo I) when target p53 gene was absent, resulting in the release of berberine and the decrease of fluorescence intensity at 537 nm. When target p53 gene was present, Cp53 DNA sequence in P1 DNA can specifically bind to p53 gene and form double-stranded DNA, so the hydrolysis of P1 DNA by Exo I was blocked, and the fluorescence intensity at 537 nm was recovered. The fluorescence of NGQDs at 440 nm does not affected from Exo I and target p53 gene, and was employed as reference. The sensing strategy showed a good linear response to p53 gene in the range of 0.2–30.0 nM with a detection limit (LOD) of 0.06 nM, and it performed well in human serum samples.

Quaternary protoberberine alkaloids and their interactions with DNA

The G-quadruplex DNA is commonly present in several protooncogenic-DNA promoters and participates in many important biological processes such as replication, transcription, and translation. Because of their supposed role in cancer, G-quadruplex DNA is often studied as a target for anti-cancer drugs. Quaternary protoberberine and tetrahydroprotoberberine alkaloids (corysamine, coptisine, stylopine), which are supposed to selectively bind these structures, have been compared in terms of stability with selected types of DNA. Influence of selected alkaloids on the stability of double-stranded DNA and non-canonical forms of DNA was observed by determining association constants of alkaloid–DNA complexes using spectroscopic methods—molecular absorption spectrometry, fluorescence spectrometry, and mass spectrometry. Furthermore, the effect of given alkaloids on the melting temperature of these DNA structures was determined using CD spectrometry. Competitive dialysis and electrospray mass spectrometry were performed for affinity comparison of certain alkaloids to different DNA structures including G-quadruplexes. These experiments have proven that corysamine and coptisine prefer interaction with G-quadruplexes in comparison to dsDNA and ssDNA, whereas tetrahydroprotoberberine alkaloid stylopine does not interact with any DNA whatsoever.

Development of Label-Free Colorimetric Assay for MERS-CoV Using Gold Nanoparticles.

Worldwide outbreaks of infectious diseases necessitate the development of rapid and accurate diagnostic methods. Colorimetric assays are a representative tool to simply identify the target molecules in specimens through color changes of an indicator (e.g., nanosized metallic particle, and dye molecules). The detection method is used to confirm the presence of biomarkers visually and measure absorbance of the colored compounds at a specific wavelength. In this study, we propose a colorimetric assay based on an extended form of double-stranded DNA (dsDNA) self-assembly shielded gold nanoparticles (AuNPs) under positive electrolyte (e.g., 0.1 M MgCl2) for detection of Middle East respiratory syndrome coronavirus (MERS-CoV). This platform is able to verify the existence of viral molecules through a localized surface plasmon resonance (LSPR) shift and color changes of AuNPs in the UV–vis wavelength range. We designed a pair of thiol-modified probes at either the 5′ end or 3′ end to organize complementary base pairs with upstream of the E protein gene (upE) and open reading frames (ORF) 1a on MERS-CoV. The dsDNA of the target and probes forms a disulfide-induced long self-assembled complex, which protects AuNPs from salt-induced aggregation and transition of optical properties. This colorimetric assay could discriminate down to 1 pmol/μL of 30 bp MERS-CoV and further be adapted for convenient on-site detection of other infectious diseases, especially in resource-limited settings.

Direct Observation of the Double-Stranded DNA Formation through Metal Ion-Mediated Base Pairing in the Nanoscale Structure.

This work demonstrates single-molecule imaging of metal-ion induced double-stranded DNA formation in DNA nanostructures. The formation of the metal ion-mediated base pairing in a DNA origami frame was examined using C-Ag-C and T-Hg-T metallo-base pairs. The target DNA strands containing consecutive C or T were incorporated into the DNA frame, and the binding was controlled by the addition of metal ions. Double-stranded DNA formation was monitored by observing the structural changes in the incorporated DNA strands using high-speed atomic force microscopy (AFM). Using the T-Hg-T base pair, the dynamic formation of unique dsDNA and its dissociation were observed. The formation of an unusual shape of dsDNA with consecutive T-Hg-T base pairs was visualized in the designed nanoscale structure.

Genome-wide mapping of nucleotide excision repair with XR-seq.

Nucleotide excision repair is a versatile mechanism to repair a variety of bulky DNA adducts. We developed excision repair sequencing (XR-seq) to study nucleotide excision repair of DNA adducts in humans, mice, Arabidopsis thaliana, yeast and Escherichia coli. In this protocol, the excised oligomers, generated in the nucleotide excision repair reaction, are isolated by cell lysis and fractionation, followed by immunoprecipitation with damage- or repair factor-specific antibodies from the non-chromatin fraction. The single-stranded excised oligomers are ligated to adapters and re-immunoprecipitated with damage-specific antibodies. The DNA damage in the excised oligomers is then reversed by enzymatic or chemical reactions before being converted into a sequencing library by PCR amplification. Alternatively, the excised oligomers containing DNA damage, especially those containing irreversible DNA damage such as benzo[a]pyrene-induced DNA adducts, can be converted to a double-stranded DNA (dsDNA) form by using appropriate translesion DNA synthesis (TLS) polymerases and then can be amplified by PCR. The current genome-wide approaches for studying repair measure the loss of damage signal with time, which limits their resolution. By contrast, an advantage of XR-seq is that the repair signal is directly detected above a background of zero. An XR-seq library using the protocol described here can be obtained in 7-9 d.

Dual-Channel Photoelectrochemical Ratiometric Aptasensor with up-Converting Nanocrystals Using Spatial-Resolved Technique on Homemade 3D Printed Device.

A near-infrared light-activated ratiometric photoelectrochemical aptasensor was fabricated for detection of carcinoembryonic antigen (CEA) coupling with upconversion nanoparticles (UCNPs)-semiconductor nanocrystals-based spatial-resolved technique on a homemade 3D printing device in which a self-regulating integrated electrode was designed for dual signal readout. The as-prepared NaYF4:Yb,Er UCNPs@CdTe nanocrystals were initially assembled on two adjacent photoelectrodes, then CEA aptamer 1 (A1) and capture DNA (CA) were modified onto two working photoelectrodes (WP1 and WP2) through covalent binding, respectively, and then gold nanoparticle-labeled CEA aptamer 2 (Au NP-A2) was immobilized on the surface of functional WP2 for the formation of double-stranded DNA. Upon target CEA introduction, the various concentrations of CEA were captured on the WP1, whereas the binding of the CEA with Au NP-A2 could be released from the WP2 thanks to the highly affinity of CEA toward A2. The dual signal readout with the "signal-off" of WP1 and "signal-on" of WP2 were employed for the spatial-resolved PEC (SR-PEC) strategy to detect CEA as an analytical model. Combining NaYF4:Yb,Er UCNPs@CdTe nanocrystals with spatial-resolved model on 3D printing device, the PEC ratiometric aptasensor based on steric hindrance effect and exciton-plasmon interactions (EPI) exhibited a linear range from 10.0 pg mL-1 to 5.0 ng mL-1 with a limit of detection of 4.8 pg mL-1 under 980 nm illumination. The SR-PEC ratiometric strategy showed acceptable stability and reproducibility with a superior anti-interference ability. This approach can provide the guidance for the design of ratiometric, multiplexed, and point-of-care biosensors.

Facile combination of beta-cyclodextrin host-guest recognition with exonuclease-assistant signal amplification for sensitive electrochemical assay of ochratoxin A

Smartly coupling exonuclease-induced target recycling signal amplifications with β-cyclodextrin host-guest recognition, a novel “signal-on” aptamer sensor for sensitive determination of ochratoxin A (OTA) was proposed for the first time. Firstly, the formation of double-strand DNA (dsDNA) was occurred by hybridizing OTA aptamer with its complementary DNA (cDNA) and as the probe DNA the cDNA at its 3′ terminal was labeled with methylene blue (MB). Next, when OTA was present, the aptamer tended to form aptamer-OTA complex with conformation of G-quadruplex instead of aptamer-cDNA duplex, leading to thus the probe DNA separating from dsDNA complex. Then the RecJf exonuclease was added, demolishing partially G-quadruplex structure and releasing a certain number of OTA. Sequentially, those released OTA would continue to react with the rest of aptamer in dsDNA, drawn into development of a new round of G-quadruplex complex, where the target cycling was realized. Meanwhile, as a signal molecule, MB modified on cDNA was liberated along with the cDNA being digested into monoucleotides by RecJf exonuclease, capable of diffusing onto the electrode surface due to host-guest recognition with β-cyclodextrin, whereupon the signal was enriched and yielded. In this way, cycles of target with continuous output of signal indicators were undergone, in which the detection of target was in return fulfilled with signal amplification owing to the joint endeavor of exonuclease and β-cyclodextrin. Under the optimal conditions, the raising signal maintained a linear relation with the logarithm of the target concentrations ranging from 10 pg/mL to 10.0 ng/mL and the detection limit reached as low as 3 pg/mL. This brand-new strategy was simple and low-cost but satisfactory in terms of detection limit, range and sensitivity, in all possibility to be applied extensively for diverse targets detection by easily alternating the corresponding aptamers.

A homogeneous DNA assay by recovering inhibited emission of rare earth ions-doped upconversion nanoparticles

Robust and easy-to-use kits specific for a particular DNA sequence are desirable for early detection of diseases. However, the major challenge with these tests is often the background fluorescence artifacts arising from biological species due to employing UV and visible range of light. Here, we have reported a near-infrared (NIR) fluorescence “turn-on” kit based on rare earth ions doped nanoparticles, upconversion nanoparticles (UCNPs), and gold nanoparticles (AuNPs), which forms a fluorescence-quencher pair, brought together by a hairpin structure through the formation of double-stranded DNA (dsDNA), with quenched upconversion luminescence. In the presence of analytes, the molecular beacon opens to push AuNPs away from UCNPs, with a distance longer than the efficient quenching distance, so that the inhibited upconversion emission will be restored. We demonstrated that this assay provides a homogeneous, facile, simple and highly selective HIV-1 based DNA detection system with restore efficiency up to 85%, and the detection limit of 5 nm.