Dye-labeled Single-stranded DNA Research Articles

Signal-On Fluorescence Biosensor for Detection of miRNA-21 Based on ROX labeled Specific Stem-Loop Probe

Background: The abnormal expression of microRNA (miRNA) influences RNA transcription and protein translation, leading to tumor progression and metastasis. Today, reliably identifying aberrant miRNA expression remains challenging, especially when employing quick, simple, and portable detection methods. Objectives: This study aimed to diagnose and detect the miR-21 biomarker with high sensitivity and specificity. Methods: Our detection approach involves immobilizing ROX dye-labeled single-stranded DNA probes (ROX-labeled ssDNA) onto MWCNTs to detect target miRNA-21. Initially, adsorbing ROX-labeled ssDNA onto MWCNTs causes fluorescence quenching of ROX. Subsequently, introducing its complementary DNA (cDNA) forms double-stranded DNA (dsDNA), which results in the desorption and release from MWCNTs, thus restoring ROX fluorescence. Results: The study examined changes in fluorescence intensities before and after hybridization with miRNA-21. The fluorescence emission intensities responded linearly to increases in miR-21 concentration from 10-9 to 3.2 × 10-6 M. The developed fluorescence sensor exhibited a detection limit of 1.12 × 10-9 M. Conclusions: This work demonstrates that using a nano-biosensor based on carbon nanotubes offers a highly sensitive method for the early detection of colorectal cancer (CRC), supplementing existing techniques.

Fluorescence Detection of miRNA-21 Using Au/Pt Bimetallic Tubular Micromotors Driven by Chemical and Surface Acoustic Wave Forces.

In this study, surface acoustic wave (SAW) systems are described for the removal of molecules that are unbound to micromotors, thereby lowering the detection limit of the cancer-related biomarker miRNA-21. For this purpose, in the first step, mass production of the Au/Pt bimetallic tubular micromotor was performed with a simple membrane template electrodeposition. The motions of catalytic Au/Pt micromotors in peroxide fuel media were analyzed under the SAW field effect. The changes in the micromotor speed were investigated depending on the type and concentration of surfactants in the presence and absence of SAW streaming. Our detection strategy was based on immobilization of probe dye-labeled single-stranded probe DNA (6-carboxyfluorescein dye-labeled-single-stranded DNA) to Au/Pt micromotors that recognize target miRNA-21. Before/after hybridization of miRNA-21 (for both w/o SAW and SAW streaming conditions), the changes in the speed of micromotors and their fluorescence intensities were studied. The response of fluorescence intensities was observed to be linearly varied with the increase of the miRNA-21 concentration from 0.5 to 5 nM under both w/o SAW and with SAW. The resulting fluorescence sensor showed a limit of detection of 0.19 nM, more than 2 folds lower compared to w/o SAW conditions. Thus, the sensor and behaviors of Au/Pt tubular micromotors were improved by acoustic removal systems.

Nucleic Acid-Gated Covalent Organic Frameworks for Cancer-Specific Imaging and Drug Release.

Developing nanoplatforms that simultaneously integrate diagnostic imaging and therapy functions has been a promising but challenging task for cancer theranostics. Herein, we report the rational design of a smart nucleic acid-gated covalent organic framework (COF) nanosystem for cancer-specific imaging and microenvironment-responsive drug release. Cy5 dye-labeled single-stranded DNA (ssDNA) for mRNA recognition was adsorbed on the surface of doxorubicin (Dox)-loaded COF nanoparticles (NPs). Dox loaded in the pores of COF NPs could strengthen the interactions between ssDNA and COF and enhance the fluorescence quenching effect toward Cy5, while the densely coated ssDNA could prevent the leakage of Dox from COF NPs. The obtained nanosystem exhibited low fluorescence signal and Dox release in normal cells; however, the ssDNA could be released by the overexpressed TK1 mRNA in cancer cells to recover the intense fluorescence signal of Cy5, and the loaded Dox could be further released for chemotherapy. Therefore, cancer cell-specific diagnostic imaging and drug release were realized with the rationally developed nanosystem. This work offers a universal nanoplatform for cancer theranostics and a promising strategy for regulating the interaction between COFs and biomolecules.

RF plasma polymer modification of graphene oxide for micromotors with improved performance

This work introduces improved performances of self-propelled tubular micromotors based on radio frequency (RF)–rotating plasma-prepared graphene oxide-poly(aniline) (GO-PANI), graphene oxide/poly(ethylaniline) (GO-PEANI), and graphene oxide/poly(fluoroaniline) (GO-PFANI) composites as outer layer and platinum (Pt) as the catalytic inner layer. Scanning electron microscopy energy-dispersive X-ray (SEM-EDX), X-ray diffraction (XRD) analysis, Fourier-transform infrared spectroscopy (FTIR), and electrochemical cyclic voltammetry (CV) techniques were utilized to characterize the RF-rotating plasma-modified graphene oxide–based composites. Incorporating the impressive features of graphene oxide (GO) and PANI or its substitute derivatives, the composite micromotors offer an efficient performance in dye-labeled single-stranded DNA immobilization due to changes in the fluorescence intensity and speed of micromotors.

Magnetically controlled 2D nano-DNA fluorescent biosensor for selective and sensitive detection of alkaline phosphatase activity

In this work, a novel magnetically controlled two-dimensional nano-DNA fluorescence sensor for sensitive and selective detection of alkaline phosphatase (ALP) activity has been developed by combining the λ exonuclease (λ exo) cleavage reaction and a δ-FeOOH nanosheet-based platform. A 5ʹ-phosphoryl-modified double-stranded DNA probe (5ʹ-P-dsDNA) was designed as a substrate for ALP, and a 5ʹ-dye-labeled single-stranded DNA/δ-FeOOH system was designed as a detection unit. In the absence of ALP, 5ʹ-P-dsDNA was degraded by λ exo to yield mononucleotides. Thus, the fluorescence of the detection unit was quenched. However, in the presence of ALP, the 5ʹ-phosphate functional group of 5ʹ-P-dsDNA was efficiently removed to form 5ʹ-hydroxyl-dsDNA, which could no longer be degraded by λ exo. Then, the dye-labeled single-stranded DNA hybridized with the 5ʹ-hydroxyl-dsDNA to form a triplex DNA (tsDNA), resulting in increased fluorescence due to the weak binding of tsDNA with the δ-FeOOH nanosheet. More importantly, a high signal-to-background ratio was obtained through magnetic separation. The detection limit for ALP was 0.02 mU/mL (3σ/S), which is much lower than that previously reported for fluorescence methods. This work revealed the application of δ-FeOOH nanosheets in the design of magnetically controlled biosensors for biomolecular detection.

DNA branched junctions induced the enhanced fluorescence recovery of FAM-labeled probes on rGO for detecting Pb2.

The reduced graphene oxide (rGO) could strongly adsorb and quench the fluorescence of dye-labeled single-stranded DNA (ssDNA); thus, it is widely applied in fluorescent sensors. However, these sensors may suffer from a limited sensitivity due to the low fluorescence recovery when adding the complementary DNA (cDNA) sequence. In this work, the powerful DNA branched junctions were constructed to improve the fluorescence recovery of FAM-labeled probe on rGO. In the presence of target Pb2+, the ribonucleotide (rA) in the substrate was cleaved specifically and the catalytic hairpin assembly of three metastable hairpins was further initiated, accompanied by the formation of DNA branched junctions. Then, the liberated Pb2+ could be recyclable. Impressively, the DNA branched junctions not only hybridize with the FAM-labeled probes with a high efficiency, but also are significantly undesirable for the rGO. Thus, a high fluorescence recovery of FAM-labeled probe on rGO was expected. The integration of the high fluorescence recovery and dual-cycle signal amplification endows the sensing strategy with a good performance for Pb2+ detection, including low detection limit (0.17nM), good selectivity, and satisfactory practical applicability. The proposed DNA branched junctions offer a novel avenue to improve the fluorescence recovery of the dye-labeled probes on rGO for biological analysis.

Biomimetic modification of silica nanoparticles for highly sensitive and ultrafast detection of DNA and Ag+ ions

Highly sensitive and selective detection of specific DNA or metal ions based on sensor platform of dye-labeled single-stranded DNA (named as ssDNA) and functional materials has attracted great research interest over the past few decades. Herein, we reported for the first time that polydopamine (PDA) modified SiO2 nanoparticles (PDA-co-SiO2 NPs) could be severed as effective fluorescent quenchers for fabrication of fluorescent DNA sensor systems for detection of functional nuclear acids and Ag+. To prepare PDA-co-SiO2 NPs, we just need mix SiO2 nanoparticles with dopamine in aqueous solution under rather mild experimental conditions. These PDA-co-SiO2 NPs-based sensor systems possess excellent sensitivity and selectivity, fast response for detection of target ssDNA and Ag+ based on different fluorescence quenching effect of PDA-co-SiO2 NPs towards ssDNA and dsDNA. Furthermore, the high fluorescence quenching ability of PDA-co-SiO2 NPs allows quantitative detection of target ssDNA with a low detection limit of 1 nM. More importantly, this method would also be easily extended for preparation of many other PDA-based materials for fabrication of fluorescent DNA sensors owing to the universal attachment of PDA with various materials and surfaces. Therefore, this work will open up a new direction for analytical detection of PDA-based functional materials.

Acoustic Nanomotors for Detection of Human Papillomavirus-Associated Head and Neck Cancer.

Human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) is a lethal disease with increasing incidence; however, technologies for early detection are limited. Nanomotors are synthetic nanostructures that can be powered by different mechanisms and functionalized for specific applications, such as biosensing. The objective of this investigation was to demonstrate an in vitro proof of concept for a novel nanomotor-based cancer detection approach toward in vivo detection of HPV-OPC. In vitro cell line incubated with ultrasound-propelled nanomotors. Basic science and engineering laboratories. Ultrasound-powered gold nanowire nanomotors were functionalized with graphene oxide and dye-labeled single-stranded DNA for the specific intracellular detection of HPV16 E6 mRNA transcripts. Nanomotors were incubated with HPV-positive or HPV-negative human OPC cells under static conditions or with an applied ultrasound field for 15 minutes. The resulting intracellular fluorescence was assessed with fluorescence microscopy and analysis software. Nanomotors incubated with RNA extracted from HPV-positive OPC cells resulted in 60.7% of maximal fluorescence recovery, while incubation with RNA extracted from HPV-negative cells produced negligible fluorescence. Nanomotor incubation with intact HPV-negative cells produced minimal fluorescence (0.01 au), while incubation with HPV-positive cells produced a detectable signal (0.43 au) under static conditions and had 2.3-times greater intensity when powered with ultrasound. Acoustically powered nanomotors can successfully identify HPV16 E6 mRNA transcripts extracellularly and within intact cells. This work represents the first step toward a novel, practical approach to address the challenge of visually detecting HPV-OPC in real time.

Ultrathin Ti3C2 nanosheets based “off-on” fluorescent nanoprobe for rapid and sensitive detection of HPV infection

MXenes as a new class of 2D materials have recently been widely applied in energy storage, electrocatalysis, sensors, adsorption, water purification, and so on, due to their tunable versatile properties. Herein, we demonstrate a simple, rapid and highly-sensitive sensing platform based on ultrathin two-dimensional MXene Ti3C2 nanosheets (Ti3C2 NSs) for selective analysis of Human papillomavirus (HPV), a major human pathogens and causative agents of cervical cancer. Ultrathin Ti3C2 NSs, obtained by exfoliating their layered HF-etched powder, exhibit high fluorescence quenching ability to dye-labeled single-stranded DNA (ssDNA) and different affinities for ssDNA and double-stranded DNA (dsDNA). Under the fluorescence quenching effect of Ti3C2 NSs, ssDNA probe (P) shows the minimal fluorescent emission. After the formation of duplex structure with its complementary target, ssDNA (T), the fluorescence intensity enhances evidently. Exonuclease III (Exo III) was used to improve the sensitivity by promoting more fluorescence enhancement. This magnified fluorescent sensor for HPV-18 detection shows a low detection limit of 100 pM and a high specificity. Furthermore, the developed DNA sensor can be employed to determine PCR amplified HPV-18 from cervical scrapes samples. It highlights ultrathin Ti3C2 NSs as a potential candidate for construction of fluorescence DNA biosensors with excellent performances.

Preparation of self-propelled Cu-Pt micromotors and their application in miRNA monitoring

Self-propelled catalytic micromotors offer considerable promise in terms of many applications. Catalytic micromotors are strongly influenced by the presence and concentration of specific ions and chemicals in the environment, making them useful as sensors and actuators. In this work, copper (Cu)-platinum (Pt) micromotors were fabricated by using the magnetron sputtering method for the first time in the literature and their applications based on the detection of miRNA-21 were evaluated. We analyzed the dependence of the mobility of Cu-Pt micromotors using different concentrations of hydrogen peroxide (H$_{2}$O$_{2})$. The presence of surfactants in the environment is also important for the movement of the micromotors. Thus, we studied the effect of three different surfactants: anionic as sodium dodecyl sulfate (SDS), cationic as cetyltrimethylammonium bromide (CTAB), and nonionic as Triton X-100. Cu-Pt micromotor motion was observed even at very low concentrations of surfactant (0.01%) and hydrogen peroxide (0.25%). miRNAs have been regarded as biomarker candidates in early diagnosis. Our sensing strategy relied on dye-labeled single-stranded DNA immobilization onto Cu-Pt micromotors that recognize the target miRNA-21. The changes in the fluorescence intensity as well as the changes in the speed of micromotors were examined before and after hybridization.

One-Step in Situ Detection of miRNA-21 Expression in Single Cancer Cells Based on Biofunctionalized MoS2 Nanosheets.

Here, we report the one-step in situ detection of targeted miRNAs expression in single living cancer cells via MoS2 nanosheet-based fluorescence on/off probes. The strategy is based on the folic acid (FA)-poly(ethylene glycol)-functionalized MoS2 nanosheets with adsorbed dye-labeled single-stranded DNA (ssDNA). Once the nanoprobes are internalized into cancer cells, the hybridization between the probes and target miRNA results in the detachment of dye-labeled ssDNA from MoS2 nanosheets surface, leading to the green fluorescence recovery. In this nanoprobe, MoS2 nanosheets offer advantages of high fluorescence quenching efficiency and extremely low toxicity. The FA conjugation could protect the probes and improve cancer cell transfection efficiency. The ability of this nanoprobe for endogenous miRNA detection in single living cancer cells is demonstrated for two types of cancer cells with different miRNA-21 expressions (MCF-7 and Hela cells). This functionalized MoS2 nanosheet-based nanoprobes could provide a sensitive and real-time detection of intracellular miRNA detection platform.

Single-Molecule Assay Development for Studying Human RNA Polymerase II Promoter-Proximal Pausing

Promoter-proximal RNA Polymerase II (Pol-II) pausing has been shown to play a significant role in transcription regulation of elongating Pol-II complexes in a large number of metazoan and mammalian genes (1). The traditional understanding of transcription regulation in mammals involved controlling Pol-II recruitment to promoters and controlling initial steps at the promoter, including pre-initiation complex formation and promoter escape. Most works investigating promoter-proximal PolII pausing have employed chromatin immunoprecipitation followed by sequencing to determine Pol-II localization or in vitro transcriptional assays using nuclear extracts analyzed with radioactive gel electrophoresis. In order to gain greater mechanistic insight into the regulation of promoter-proximal Pol-II pausing, we use single molecule ALEX spectroscopy to monitor RNA transcripts production as function of composition and order of addition of transcription factors to an in vitro reconstituted human Pol-II system. The RNA transcripts are detected by complementary doubly dye-labeled single-stranded DNA (ssDNA) probes. The human gene HSPA1B for heat shock protein 70 (Hsp70) is used as a model system due to its extensive characterization in drosophila. Our approach provides a rapid, sensitive and robust avenue for screening protein factors regulating promoter-proximal Pol-II pausing.1. H. Kwak, J. T. Lis, Control of transcriptional elongation. Annu. Rev. Genet. 47, 483-508 (2013).

Single Cell Real-Time miRNAs Sensing Based on Nanomotors.

A nanomotor-based strategy for rapid single-step intracellular biosensing of a target miRNA, expressed in intact cancer cells, at the single cell level is described. The new concept relies on the use of ultrasound (US) propelled dye-labeled single-stranded DNA (ssDNA)/graphene-oxide (GO) coated gold nanowires (AuNWs) capable of penetrating intact cancer cells. Once the nanomotor is internalized into the cell, the quenched fluorescence signal (produced by the π-π interaction between GO and a dye-labeled ssDNA) is recovered due to the displacement of the dye-ssDNA probe from the motor GO-quenching surface upon binding with the target miRNA-21, leading to an attractive intracellular "OFF-ON" fluorescence switching. The faster internalization process of the US-powered nanomotors and their rapid movement into the cells increase the likelihood of probe-target contacts, leading to a highly efficient and rapid hybridization. The ability of the nanomotor-based method to screen cancer cells based on the endogenous content of the target miRNA has been demonstrated by measuring the fluorescence signal in two types of cancer cells (MCF-7 and HeLa) with significantly different miRNA-21 expression levels. This single-step, motor-based miRNAs sensing approach enables rapid "on the move" specific detection of the target miRNA-21, even in single cells with an extremely low endogenous miRNA-21 content, allowing precise and real-time monitoring of intracellular miRNA expression.

Graphitic carbon nitride nanosheet-based multicolour fluorescent nanoprobe for multiplexed analysis of DNA

We demonstrate that nanosheets composed of graphitic carbon nitride (g-C3N4) can serve as a low-cost and efficient fluorescent nanoprobe for the multiplexed detection of DNA in solution. The strategy is based on the finding that g-C3N4 is capable of binding dye-labeled single-stranded DNA (ssDNA) which results in quenching of the fluorescence of the dye. If target DNA hybridizes with dye-labeled ssDNA, the interaction between dye-labeled ssDNA and g-C3N4 is weakened, and this results in desorption of the dsDNA from the surface of the g-C3N4 and in recovery of fluorescence. The large surface area of g-C3N4 nanosheets allows for simultaneous quenching of multicolor DNA probes labeled with different dyes, leading to the development of multiplexed DNA sensors for the detection of multiple DNA targets in a single solution. By using one 15-mer DNA fragment and one 18-mer DNA fragment as proof-of-principle analytes, the method displayed good analytical performance. The limits of detection are 75 and 62 pM, respectively. The method is simple and sensitive, and was used to detect DNA in serum samples. We perceive that this method represents a new approach towards multiplexed assays for applications in DNA monitoring, clinical diagnosis, and in the detection of genetic disorders.

A novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor for sensitive detection of prostate specific antigen.

Prostate specific antigen (PSA) is a significant and the most widely used biomarker for the early diagnosis of prostate cancer and its subsequent treatment. A MoS2 nanosheet is a two-dimensional (2D) layered nanomaterial analogous to graphene. However, a MoS2 nanosheet has a higher fluorescence-quenching ability than graphene when applied to a dye-labeled single-stranded DNA probe. In this work, we propose a novel aptamer-functionalized MoS2 nanosheet fluorescent biosensor that detects PSA. The binding of the aptamer to the target PSA induces a rigid aptamer structure which makes the integration with the MoS2 nanosheet very weak. This results in the release of the aptamer probe from the nanosheet surface and restores the quenched fluorescence. This approach has the advantage of simple design and rapid detection of PSA. The biosensor has the merits of high sensitivity and high selectivity with a detection limit for the PSA of 0.2ng/mL. The biosensor was also successfully applied to the detection of PSA in human serum samples with satisfactory results. The foregoing indicates its promising application to real-life biological samples.

A barium based coordination polymer for the activity assay of deoxyribonuclease I.

A new coordination polymer which shows an unusual 2D inorganic connectivity was constructed. This compound exhibits distinct fluorescence quenching ability to the dye-labeled single-stranded DNA probes with different lengths, based on which an analytical method was developed for the activity assay of deoxyribonuclease I.

Highly selective and sensitive nucleic acid detection based on polysaccharide-functionalized silver nanoparticles

Polysaccharide-functionalized silver nanoparticles (Oc-AgNPs) with a mean diameter of 15nm were utilized as a novel and effective fluorescence-sensing platform for nucleic acid detection. Tests on the oligonucleotide sequences associated with the human immunodeficiency virus as a model system showed that the Oc-AgNPs effectively absorbed and quenched dye-labeled single-stranded DNA through strong hydrogen bonding interactions and slight electrostatic attractive interactions. The proposed system efficiently differentiated between complementary and mismatched nucleic acid sequences with high selectivity and good reproducibility at room temperature.

Magnified fluorescence detection of silver(I) ion in aqueous solutions by using nano-graphite-DNA hybrid and DNase I

This paper describes a novel approach utilizing nano-graphite-DNA hybrid and DNase I for the amplified detection of silver(I) ion in aqueous solutions for the first time. Nano-graphite can effectively quench the fluorescence of dye-labeled cytosine-rich single-stranded DNA due to its strong π–π stacking interactions; however, in the presence of Ag+, C–Ag+–C coordination induces the probe to fold into a hairpin structure, which does not adsorb on the surface of nano-graphite and thus retains the dye fluorescence. Meanwhile, the hairpin structure can be cleaved by DNase I, and in such case Ag+ is delivered from the complex. The released Ag+ then binds other dye-labeled single-stranded DNA on the nano-graphite surface, and touches off another target recycling, resulting in the successive release of dye-labeled single-stranded DNA from the nano-graphite, which leads to significant amplification of the signal. The present magnification sensing system exhibits high sensitivity toward Ag+ with a limit of detection of 0.3nM (S/N=3), which is much lower than the standard for Ag+ in drinking water recommended by the Environmental Protection Agency (EPA). The selectivity of the sensor for Ag+ against other biologically and environmentally related metal ions is outstanding due to the high specificity of C–Ag+–C formation. Moreover, the sensing system is used for the determination of Ag+ in river water samples with satisfying results. The proposed assay is simple, cost-effective, and might open the door for the development of new assays for other metal ions or biomolecules.

Applications of porous organic frameworks (POFs) in detection of nucleic acid and exonuclease I activity.

As a two-dimensional (2D) ordered porous organic framework (POF), PAF-6 is demonstrated to have an extraordinarily high fluorescence quenching ability to dye-labeled single-stranded DNA (ssDNA). Based on its different affinities to ssDNA and double-stranded DNA (dsDNA), and to ssDNAs with different lengths, PAF-6 is firstly utilized as a simple, cost-efficient, sensitive and selective sensing platform for sequence-specific detection of DNA and activity analysis of exonuclease I (Exo I). In these two systems, the sensing approach is accomplished by simply mixing the dye-labeled ssDNA probe with the targets and PAF-6. The targets of DNA and Exo I are specifically and sensitively detected with detection limits of 0.6 nM and 0.03 U mL-1 (S/N = 3), respectively, by using PAF-6 as a fluorescence quencher of the dye-labeled ssDNA probe. The results of this study suggest that PAF-6 can be developed as an excellent platform for the detection of nucleic acid and nuclease activity. In addition, PAF-6 exhibits a remarkable ability to protect ssDNA probe from enzymatic digestion, which may greatly extend the applications of the proposed ssDNA probe/PAF-6 sensing system to bioanalysis and biomedicine.

SERS Primers and Their Mode of Action for Pathogen DNA Detection

SERS primers have been used to directly detect specific PCR products utilizing the difference in adsorption of single-stranded and double-stranded DNA onto nanoparticle surfaces. Herein, seven parameters important for improved positive SERS assays for real applications were investigated. First, we applied a model system for optimization experiments, followed by a PCR assay to detect pathogen DNA, and then the introduction of a new assay that utilizes the 5'→3' exonuclease activity of Taq DNA polymerase to partly digest the SERS probe, generating dye-labeled single-stranded DNA increasing the SERS signals for detection of pathogen DNA. Applying the model system, it was found that uni-molecular SERS primers perform better than bi-molecular SERS primers. However, within the PCR assays, it was found that uni- and bi-molecular SERS primers performed very similarly, and the most reproducible results were obtained using the 5'→3' exonuclease digestion assay. These SERS-based assays offer new routes over conventional fluorescence-based techniques without compromising sensitivity or selectivity.