Detection Of Nucleic Acid Targets Research Articles

Visible-light triggered templated ligation on surface using furan-modified PNAs.

Oligonucleotide-templated reactions are frequently exploited for target detection in biosensors and for the construction of DNA-based materials and probes in nanotechnology. However, the translation of the specifically used template chemistry from solution to surfaces, with the final aim of achieving highly selective high-throughput systems, has been difficult to reach and therefore, poorly explored. Here, we show the first example of a visible light-triggered templated ligation on a surface, employing furan-modified peptide nucleic acids (PNAs). Tailored photo-oxidation of the pro-reactive furan moiety is ensured by the simultaneous introduction of a weak photosensitizer as well as a nucleophilic moiety in the reacting PNA strand. This allows one to ensure a localized production of singlet oxygen for furan activation, which is not affected by probe dilution or reducing conditions. Simple white light irradiation in combination with target-induced proximity between reactive functionalities upon recognition of a short 22mer DNA or RNA sequence that functions as a template, allows sensitive detection of nucleic acid targets in a 96 well plate format.

Smartphone-Based Droplet Digital LAMP Device with Rapid Nucleic Acid Isolation for Highly Sensitive Point-of-Care Detection.

While advances in microfluidics have enabled rapid and highly integrated detection of nucleic acid targets, the detection sensitivity is still unsatisfactory in the current POC (point-of-care) detection systems, especially for low abundance samples. In this study, a chip that integrates rapid nucleic acid extraction based on IFAST (immiscible phase filtration assisted by surface tension) and digital isothermal detection was developed to achieve highly sensitive POC detection within 60 min. Based on the interface theory, the factors influencing the interface stability of the IFAST process were studied, and the IFAST nucleic acid extraction conditions were optimized to increase the nucleic acid extraction recovery rate to 75%. Spiral mixing channel and flow-focusing droplet generation structure were designed to achieve the mixing and sample partitioning by applying negative pressure. A portable microdroplet fluorescence detection device was developed based on smartphone imaging. Validation tests were carried out for quantification of low-abundance cfDNA and detection of mutations.

Three-way junction-induced isothermal amplification for nucleic acid detection

We, herein, describe a three-way junction (3WJ)-induced isothermal amplification (ThIsAmp) reaction for target nucleic acid detection. In this strategy, target nucleic acid induces the formation of 3WJ structure by associating a specially designed ThIsAmp template and ThIsAmp primer. Upon the formation of 3WJ structure, ThIsAmp primer is subjected to continuously repeated extension and nicking reaction by the combined activities of DNA polymerase and nicking endonuclease, consequently producing a large number of trigger strands. The trigger strands then initiate two separate but interconnected pathways by binding to either 3’ overhang of ThIsAmp template within the 3WJ structure or free ThIsAmp template. As a consequence, a large number of final double-stranded DNA products are produced under an isothermal condition, which can be monitored in real-time by detecting the fluorescence intensity resulting from SYBR Green I staining. Based on this principle, we successfully detected target DNA down to 78.1 aM with excellent specificity. The sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling target nucleic acid detection.

Solid-phase PCR based on thermostable, encoded magnetic microspheres for simple, highly sensitive and multiplexed nucleic acid detection

Multiplexed detection of nucleic acids is important for pathogen identification and disease diagnosis. Though real-time quantitative PCR allows for simultaneous amplification of different genes in a single test, the interferences between various primers inevitably cause difficulty in assay design, and pose limitation on multiplexing degree. Conventional solid-phase PCR (SP-PCR) on plenary microarrays can assess more targets than real-time quantitative PCR. However, they are suffering from low reaction efficiency, high background noise, and requiring special equipment and expertise for reliable analysis. Here, we addressed the challenges by performing multiplex PCR on thermostable, tailor-made host-guest encoded magnetic microspheres (Beads based PCR, BB-PCR), which were constructed by coupling magnetic host microspheres with fluorescent guest nanoparticles. As proof of concept, a 3-plex barcoded microbeads with high thermostability and low non-specific fluorescence absorption were selected as model BB-PCR carriers to amplify and detect three gene targets in Salmonella Enteritidis. After BB-PCR reaction, the barcoding and reporting signals were easily identified through flow cytometry. Attributed to the effective interactions between reagents and microspheres during PCR reaction, this method showed high sensitivity of 10 copies/reaction, which was comparable to that of liquid PCR. Thus, BB-PCR developed here allowed for simple, rapid, highly sensitive and high throughput detection of nucleic acid targets, which paves the way for routine multiplexed molecular diagnostics in clinical laboratories.

Subzymes: Regulating DNAzymes for point of care nucleic acid sensing

Rapid, sensitive and affordable nucleic acid sensors that can operate in a point of care (POC) setting are highly desired; however, their availability and implementation remain limited. We report the development of a strategy for building sensors which use novel catalytic nucleic acid structures, herein referred to as ‘Subzymes’. We examine the effectiveness of Subzymes to mediate signal amplification in a universal manner allowing faster rates of detection. We created Subzymes by manufacturing composite oligonucleotides that contain a catalytic nucleic acid component and a substrate component. We demonstrate that the activity of some catalytic DNAzyme components can be inhibited by attaching the Subzymes to micro-particles. Subsequent cleavage of a Subzyme’s internal substrate results in the release and activation of the surface-bound DNAzyme, thus providing a mechanism to control the catalytic activity of the DNAzyme. We demonstrate that released DNAzymes are capable of cleaving fluorescent-labelled reporter substrates to generate a signal, thus confirming the restoration of their catalytic activity. The addition of Subzymes to reactions where the detection of a target is achieved by multi-component nucleic acid enzymes, known as PlexZymes, showed improved target sensitivities in a shorter amount of time (10 pM of target detected in under 60 min), demonstrating rapid detection of nucleic acid targets without the use of protein-enzymes. Subzymes are constructed from low-cost materials and operate under isothermal reaction conditions which are advantageous for on-site diagnostic testing. Thus, Subzymes offer a universal, rapid and affordable tool for nucleic acid sensing, providing new avenues for POC testing.

Inverse Molecular Sentinel-Integrated Fiberoptic Sensor for Direct and in Situ Detection of miRNA Targets.

Molecular advances have been made in analysis systems for a wide variety of applications ranging from biodiagnostics, biosafety, bioengineering, and biofuel research applications. There are, however, limited practical tools necessary for in situ and accurate detection of nucleic acid targets during field work. New technology is needed to translate these molecular advances from laboratory settings into the real-life practical monitoring realm. The exquisite characteristics (e.g., sensitivity and adaptability) of plasmonic nanosensors have made them attractive candidates for field-ready sensing applications. Herein, we have developed a fiber-based plasmonic sensor capable of direct detection (i.e., no washing steps required) of nucleic acid targets, which can be detected simply by immerging the sensor in the sample solution. This sensor is composed of an optical fiber that is decorated with plasmonic nanoprobes based on silver-coated gold nanostars (AuNS@Ag) to detect target nucleic acids using the surface-enhanced Raman scattering (SERS) sensing mechanism of nanoprobes referred to as inverse molecular sentinels (iMS). These fiber-optrodes can be reused for several detection-regeneration cycles (>6). The usefulness and applicability of the iMS fiber-sensors was tested by detecting target miRNA in extracts from leaves of plants that were induced to have different expression levels of miRNA targets. These fiber-optrodes enable direct detection of miRNA in plant tissue extract without the need for complex assays by simply immersing the fiber in the sample solution. The results indicate the fiber-based sensors developed herein have the potential to be a powerful tool for field and in situ analysis of nucleic acid samples.

Flap endonuclease-initiated enzymatic repairing amplification for ultrasensitive detection of target nucleic acids.

A new isothermal nucleic acid amplification method termed FERA (Flap endonuclease-initiated Enzymatic Repairing Amplification) is developed for the ultrasensitive detection of target nucleic acids. In the FERA method, flap endonuclease (FEN) catalyzes the hydrolytic cleavage at the junction of single- and double-stranded DNAs which is formed only in the presence of target nucleic acids, and releases short oligonucleotides to promote the cyclic enzymatic repairing amplification (ERA) combined with FEN-based amplification. As a result, a large amount of single- and double-stranded DNAs are generated under the isothermal conditions, leading to the high fluorescence intensity from the SYBR I green dye. Relying on the powerful amplification method, we successfully determined the target nucleic acids with a limit of detection as low as 15.16 aM, which corresponds to approximately 180 molecules in 20 μL reaction volume, and verified the practical applicability by detecting long target nucleic acids derived from Chlamydia trachomatis.

Rapid and Sensitive Detection of Citrus tristeza virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay.

Loop-mediated isothermal amplification (LAMP) is one recently developed gene amplification technique that emerges as a simple and quick diagnostic tool for early detection of nucleic acid targets. The LAMP technique works on the principle of strand displacement activity of Bst polymerase. It contains a set of four specially designed primers, which recognizes six different regions on the target nucleotide sequence. In the LAMP reaction, amplification is carried out in an isothermal conditions (60-65°C) using simple and inexpensive device like water bath or dry bath. Additional benefits of LAMP technique are that final results can be seen directly with naked eyes by adding intercalating dye SYBR Green I in the reaction tube. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is one of the novel techniques used for detection of RNA targets. The technology has been successfully applied for rapid and sensitive detection of Citrus tristeza virus (CTV) by using four oligo-primers, targeting a conserved coat protein gene (CPG) of an Indian CTV isolate. The result of assay is visible in naked eyes easily in the presence of SYBR Green I (100×) or on 1.5% agarose gel electrophoresis. CTV-RT-LAMP could be used away from plant pathology laboratories even in remote location.

Visual/CVG-AFS/ICP-MS multi-mode and label-free detection of target nucleic acids based on a selective cation exchange reaction and enzyme-free strand displacement amplification.

Conventional atomic spectrometry biosensors usually require labeling and separation of signaling molecules. Visual assays have direct and effective characteristics; however, they have poor accuracy. We intended to improve the analytical performance of our previous work and simplify the experimental operation while maintaining the advantages of simple operation and low cost. Herein, we describe the development of a visual, chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS) and inductively coupled plasma-mass spectrometry (ICP-MS) three-mode method for the analysis of nucleic acids via CdTe quantum dot (QD)-assisted selective cation exchange reaction and enzyme-free strand displacement amplification. This work mainly utilized the ability of CdTe QDs to selectively differentiate free Hg2+ from the T-Hg2+-T complex in addition to the simple selective membrane filtration separation of Cd2+ from CdTe QDs to improve the performance of label-free bioassay methods. Due to the superior optical features of CdTe QDs, they can not only be used as a signal molecule for atomic spectroscopy, but also for direct use in visual readings. Under optimal experimental conditions, the developed strategy displayed a wide linear range along with limits of detection (LODs) of 10 fM and 3 fM (2 fM) in the linear concentration ranges of 10 fM-100 pM and 10 fM-1 nM with the naked eye and CVG-AFS (ICP-MS) assays, respectively. This method also exhibited excellent DNA sequence specificity. This assay had advantageous characteristics such as an easy operation, simple design, high sensitivity, and diversified signal readout manner, which demonstrate its great potential in medical diagnosis applications.

DNA engineered micromotors powered by metal nanoparticles for motion based cellphone diagnostics

HIV-1 infection is a major health threat in both developed and developing countries. The integration of mobile health approaches and bioengineered catalytic motors can allow the development of sensitive and portable technologies for HIV-1 management. Here, we report a platform that integrates cellphone-based optical sensing, loop-mediated isothermal DNA amplification and micromotor motion for molecular detection of HIV-1. The presence of HIV-1 RNA in a sample results in the formation of large-sized amplicons that reduce the motion of motors. The change in the motors motion can be accurately measured using a cellphone system as the biomarker for target nucleic acid detection. The presented platform allows the qualitative detection of HIV-1 (n = 54) with 99.1% specificity and 94.6% sensitivity at a clinically relevant threshold value of 1000 virus particles/ml. The cellphone system has the potential to enable the development of rapid and low-cost diagnostics for viruses and other infectious diseases.

Ratiometric Fluorescence Coding for Multiplex Nucleic Acid Amplification Testing.

Although nucleic acid amplification testing (NAAT) has become the cornerstone for molecular diagnosis of diseases, expanding the multiplexed detection capacity of NAAT remains an important objective. To this end, encoding each nucleic acid target with a specific fluorescently labeled probe has been the most mature approach for multiplexed detection. Unfortunately, the number of targets that can be differentiated via this one-target-one-fluorophore multiplexed detection approach is restricted by spectral overlaps between fluorophores. In response, we present herein a new multiplexed detection approach termed ratiometric fluorescence coding, in which we encode each nucleic acid target with a specific ratio between two standard fluorophores. In ratiometric fluorescence coding, we employ the padlock probe chemistry to encode each nucleic acid target with a specific number of binding sites for two probes labeled with different fluorophores. Coupling the padlock probes with either rolling circle amplification (RCA) or hyperbranched rolling circle amplification (HRCA), we transform each nucleic acid target into a specific template that allows hybridization with the fluorescently labeled probes at predesigned ratios, thereby achieving multiplexed detection. For demonstration, we detected DNA targets from six infectious diseases and demonstrated the potential for further expanding the multiplexing capability of our approach. With further development, ratiometric fluorescence coding has the potential to enable highly multiplexed detection of nucleic acid targets and facilitate molecular diagnosis of diseases.

Probing DNA Hybridization Equilibrium by Cationic Conjugated Polymer for Highly Selective Detection and Imaging of Single-Nucleotide Mutation.

Hybridization-based probes emerge as a promising tool for nucleic acid target detection and imaging. However, the single-nucleotide selectivity is still challenging because the specificity of hybridization reaction is typically low at room temperature. We disclose an effective and simple method for highly selective detection and in situ imaging of single-nucleotide mutation (SNM) by taking the advantages of the specific hybridization of short duplex and the signal amplifying effect of cationic conjugated polymer (CCP). Excellent discrimination of the nucleic acid strands only differing by single nucleotide was achieved enabling the sensitive detection of SNM at the abundance as low as 0.1%. Single-molecule fluorescence resonance energy transfer (smFRET) study reveals that the presence of CCP enhances the perfect matched duplex and the mismatched duplex to a different extent, which can be an explanation for the high single-nucleotide selectivity. Due to the simple design of the probe and the stable brightness of CCP, highly selective mRNA in situ imaging was achieved in fixed cells. Melanoma cell line A375 with BRAF V600E point mutation exhibits higher FRET efficiency than liver cancer cell line HegG2 that was not reported having the mutation at this point.

Surface-enhanced Raman scattering nanosensors for in vivo detection of nucleic acid targets in a large animal model

Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo detection remains very challenging. Here, we demonstrated the proof-of-principle of in vivo detection of nucleic acid targets using a promising type of surface-enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo nanosensor used in this study involves the “inverse molecular sentinel” detection scheme using plasmonics-active nanostars, which have tunable absorption bands in the near infrared region of the “tissue optical window”, rendering them efficient as an optical sensing platform for in vivo optical detection. Ex vivo measurements were also performed using human skin grafts to demonstrate the detection of SERS nanosensors through tissue. In this study, a new core–shell nanorattle probe with Raman reporters trapped between the core and shell was utilized as an internal standard system for self-calibration. These results illustrate the usefulness and translational potential of the SERS nanosensor for in vivo biosensing.

Evaluation of an Assay Based On Multiple Detection Temperature Technique for Simultaneous Detection of Viral Gastroenteritis-Causing Pathogens

Background: Multiple detection temperature (MuDT) technique is an advanced method for the analysis of multiple Ct (cycle threshold) values in a single channel. Objectives: The advantage of this method has been shown only in DNA samples, restricting its diagnostic applicability. This technique was evaluated in this study for its efficacy in the analysis of target RNA. Methods: Allplex GI-virus assay was developed to detect pathogens causing viral gastroenteritis, one of the major diseases caused by RNA viruses. This one-step multiplex real-time polymerase chain reaction (PCR) based on the MuDT technique permits simultaneous amplification and detection of target nucleic acids of norovirus GI, norovirus GII, rotavirus A, adenovirus F, astrovirus, and sapovirus genogroups. The assay was tested for analytical sensitivity, cross-reactivity, repeatability, and applicability to clinical samples. Results: The analytical performance was validated for each target. The assay demonstrated high analytical sensitivity and no cross-reactivity, and the repeatability tests showed excellent performance with high accuracy. Analytical performance validation indicated high positive agreement and negative agreement for this method. In the analyses comparing Allplex GI-virus Assay and commercial Seeplex Diarrhea-V ACE Detection using clinical specimens, the positive and negative agreements between the test results were found to be 94.9% and 98.8%, respectively. Statistical analysis showed that there was no difference in the performance between the two products. Conclusions: The Allplex GI-virus Assay can rapidly detect six viruses in a single tube without the complementary DNA synthesis step, and this assay was shown to represent an improved molecular diagnostic tool for the simultaneous detection of several RNA viruses. Therefore, our results suggest that the MuDT technique may represent a new molecular diagnostic method for the detection of RNA viruses.

Detection of Specific Nucleic Acids Utilizing DNA Nano-Tweezers Structures

We have developed signal-transducing molecules for detecting specific nucleic acids in a homogeneous assay format. A DNA nano-tweezers (DNA-NT) structure, self-assembled from three single-stranded DNAs, was employed as a main component that alters its structure from an open state to a closed state in recognition of target nucleic acids. This dynamic structural change was utilized for generating a target-specific sensing signal. A fluorescence resonance energy transfer (FRET)-paired fluorescent dyes were modified onto a DNA-NT to generate a target-specific sensing signal. This FRET-based DNA-NT alters its structure in response to target nucleic acids, resulting in the closing of the distal relation of FRET-paired fluorescent dyes. The FRET-based DNA-NT generates the FRET signal only when it recognizes target nucleic acids, and thus the target detection can be performed in a homogeneous assay format, which does not require washing procedure. Therefore, when the FRET-based DNA-NT is introduced into a cell, the continuous detection of specific target mRNA inside living cells can be realized in a non-destructive manner. Live-cell imaging analysis of drug stimulated cells with the FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those of target mRNA measured with the conventional mass analysis technique of semi-quantitative real-time polymerase chain reaction. Thus, it was concluded that continuous monitoring of specific genes is feasible with a FRET-based DNA-NT, even in a single cell manner. As another example of a component for signal generation, a G-quadruplex (Gq) composed a guanine-rich DNA sequence was employed. A Gq is one of the well-characterized DNazymes that exhibits a peroxidase activity with the help of hemin as a co-factor. In our design, the split Gq sequences were added to both ends of a DNA-NT instead of the FRET-paired fluorescent dyes. This split-Gq-based DNA-NT alters its structure from an open state to a closed state, inducing the complementation of the split Gq that exhibits restored peroxidase activity. A model target RNA was specifically recognized by the split Gq-based DNA-NT. The peroxidase activity was measured by following the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), which gave a greenish colorimetric response, and was proportional to the concentration of model target RNA. The split-Gq-based DNA-NT generates a peroxidase activity only when it recognizes target nucleic acids, and thus the target detection can be performed in a homogeneous assay format that is suitable for a portable format. Therefore, the detection of specific nucleic acids with the split Gq-based DNA-NT has great potential for use in a wide variety of applications, such as point-of-care testing and point-of-need testing. The DNA-NT-based signal-transducing molecules, enabling the detection of target nucleic acids in a homogeneous assay format, recognize the target without competitive reactions in a bimolecular reaction and directly produce a readily detectable signal. Notably, the target recognition sites of DNA-NT-based signal-transducing molecules can easily be designed without delicate optimization of tweezers structure. Therefore, the DNA-NT-based signal-transducing molecules are expected to become a versatile tool for detecting specific nucleic acids.

Rolling circle amplification as isothermal gene amplification in molecular diagnostics.

Rolling circle amplification (RCA) developed in the mid-1990s has been widely used as an efficient isothermal DNA amplification process for molecular diagnosis. This enzymatic process amplifies target DNA sequences with high fidelity and specificity by using the strand displacing DNA polymerases. The product of RCA is long single-stranded DNA that contains tandem repeat of target sequence. Isothermal reaction amplification condition of RCA has an advantage over conventional polymerase chain reaction, because no temperature cycling devices are needed for RCA. Thus, RCA is suitable tool for point-of-care detection of target nucleic acids as well as facile detection of target genes. Combined with various detection methods, RCA could amplify and detect femtomolar scale of target nucleic acids with a specificity of one or two base discrimination. Herein, RCA technology is reviewed with an emphasis on molecular diagnosis of microRNAs, infectious pathogens, and point mutations.

Adsorption and isolation of nucleic acids on cellulose magnetic beads using a three-dimensional printed microfluidic chip

While advances in genomics have enabled sensitive and highly parallel detection of nucleic acid targets, the isolation and extraction of the nucleic acids remain a critical bottleneck in the workflow. We present here a simple 3D printed microfluidic chip that allows for the vortex and centrifugation free extraction of nucleic acids. This novel microfluidic chip utilizes the presence of a water and oil interface to filter out the lysate contaminants. The pure nucleic acids, while bound on cellulose particles, are magnetically moved across the oil layer. We demonstrated efficient and rapid extraction of spiked Human Papillomavirus (HPV) 18 plasmids in specimen transport medium, in under 15 min. An overall extraction efficiency of 61% is observed across a range of HPV plasmid concentrations (5 × 10(1) to 5 × 10(6) copies/100 μl). The magnetic, interfacial, and viscous drag forces inside the microgeometries of the chip are modeled. We have also developed a kinetics model for the adsorption of nucleic acids on cellulose functionalized superparamagnetic beads. We also clarify here the role of carrier nucleic acids in the adsorption and isolation of nucleic acids. Based on the various mechanistic insights detailed here, customized microfluidic devices can be designed to meet the range of current and emerging point of care diagnostics needs.

Intelligent DNA machine for the ultrasensitive colorimetric detection of nucleic acids.

As DNA is employed to serve as a smart building block, an increasing interest has been devoted to the development of different DNA-based machines for the specific purpose, for example, the exploration of inter- or intramolecular interaction. In the current contribution, we developed an intelligent DNA machine and its operation can be designed to execute the ultrasensitive colorimetric detection of target nucleic acids. The DNA machine consists of a hairpin probe (HP) and an assistant template (AT). Using p53 gene as the target model to trigger the molecular machine operation, cyclic nucleic acid strand displacement polymerization (CNDP) was specifically induced, leading to the DNAzyme mediated catalytic reaction for signal readout. Specifically, with the help of polymerase and nickase, one target molecule was able to drive DNA nano-mechanical devices one-by-one through the hybridization/polymerization displacement cycles, and every initiated machine continued to operate, causing the dramatic accumulation of G-quadruplex-contained products. The G-quadruplex structure after binding to hemin could act as a horseradish peroxidase (HRP)-mimicking DNAzyme and catalyzed the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by H2O2. As a result, an enhanced color change could be detected because of the generation of oxidation product ABTS•(+). In this way, the DNA machine has no any signal loss and enables the quantitative measurement of p53 DNA with a detection limit of 10fM, indicating great promise for unique application in biomedical research and early clinical diagnosis.

Formation of Silver Nanostructures by Rolling Circle Amplification Using Boranephosphonate-Modified Nucleotides.

We investigate the efficiency of incorporation of boranephosphonate-modified nucleotides by phi29 DNA polymerase and present a simple method for forming large defined silver nanostructures by rolling circle amplification (RCA) using boranephosphonate internucleotide linkages. RCA is a linear DNA amplification technique that can use specifically circularized DNA probes for detection of target nucleic acids and proteins. The resulting product is a collapsed single-stranded DNA molecule with tandem repeats of the DNA probe. By substituting each of the natural nucleotides with the corresponding 5'-(α-P-borano)deoxynucleosidetriphosphate, only a small reduction in amplification rate is observed. Also, by substituting all four natural nucleotides, it is possible to enzymatically synthesize a micrometer-sized, single-stranded DNA molecule with only boranephosphonate internucleotide linkages. Well-defined silver particles are then readily formed along the rolling circle product.

Label-free molecular beacons for biomolecular detection.

Biomolecular detection and imaging methods provide quantitative measurements essential for biological research. In this context, molecular beacon based sensors have emerged as powerful, no-wash imaging agents, providing target-specific fluorescent activation for nucleic acids, proteins, and small molecules. Conventional molecular beacons require double-labeled DNA sequences, which are costly and time-consuming to prepare. To address this issue, we developed DNA based label-free molecular beacons consisting of two regions: a signal-generating region based on human telomeric G-quadruplex sequence that activates Thioflavin T fluorescence and a target recognition sequence designed to interact in a molecular beacon format. We demonstrated the utility of these probes for the selective detection of DNA, RNA, and protein. Multiple probes were applied against a single target to achieve improved brightness in fluorescence detection of nucleic acid targets. This label-free strategy provides a straightforward, cost-effective alternative to fluorescently labeled oligonucleotides in biomolecular detection and imaging.