ssDNA Sequences Research Articles

Fast and Ultrasensitive Detection of Monkeypox by a Pyrococcus furiosus Argonaute System Coupled with a Short Amplification.

Monkeypox virus (MPXV), the pathogen responsible for the infectious disease monkeypox, causes lesions on the skin, lymphadenopathy, and fever. It has posed a global public health threat since May 2022. Highly sensitive and specific detection of MPXV is crucial for preventing the spread of the disease. Pyrococcus furiosus Argonaute (PfAgo) is an artificial DNA-guided restriction cleavage enzyme programmable with 5'-phosphorylated ssDNA sequences, which can be developed to specifically detect nucleic acids of pathogens. Here, a PfAgo-based system was established for the detection of MPXV-specific DNA targeting the F3L gene. A short amplicon of 79 bp could be obtained through a fast PCR procedure, which was completed within 45 min. Two 5'-phosphorylation guide DNAs were designed to guide PfAgo to cleave the amplicon to obtain an 18 bp 5'-phosphorylation sequence specific to MPXV, not to other orthopoxviruses (cowpox, variola, and vaccinia viruses). The 18 bp sequence guided PfAgo to cleave a designed probe specific to MPXV to emit fluorescence. With optimized conditions for the PfAgo-MPXV system, it could be completed in 60 min for the detection of the extracted MPXV DNA with the limit of detection (LOD) of 1.1 copies/reaction and did not depend on expensive instruments. Successful application of the PfAgo-MPXV system in sensitively detecting MPXV in simulated throat swabs, skin swabs, sera, and wastewater demonstrated the system's good performance. The PfAgo platform, with high sensitivity and specificity established here, has the potential to prevent the spread of MPXV.

A rapid VEGF-gene-sequence photoluminescence detector for osteoarthritis.

Osteoarthritis (OA) has become a serious problem to the human society for years due to its high economic burden, disability, pain, and severe impact on the patient's lifestyle. The importance of current clinical imaging modalities in the assessment of the onset and progression of OA is well recognized by clinicians, but these modalities can only detect OA in the II stage with significant structural deterioration and clinical symptoms. Blood vessel formation induced by vascular endothelial growth factor (VEGF) occurs in the early stage and throughout the entire course of OA, enables VEGF relating gene sequence to act as a biomarker in the field of early diagnosis and monitoring of the disease. Here in, a facile rapid detection of VEGF relating ssDNA sequence was developed, in which manganese-based zeolitic imidazolate framework nanoparticles (Mn-ZIF-NPs) were synthesized by a simple coprecipitation strategy, followed by the introduction and surficial absorption of probe ssDNAs and the CRISPR/Cas12a system components. Furthermore, fluorescence experiments demonstrated that the biosensor displayed a low detection limit of 2.49nM, a good linear response to the target ssDNA ranging from 10nM to 500nM, and the ability of distinguishing single nucleotide polymorphism. This finding opens a new window for the feasible and rapid detection of ssDNA molecules for the early diagnose of OA.

CRISPR-Cas12a based target recognition initiated duplex-specific nuclease enhanced fluorescence and colorimetric analysis of cell-free DNA (cfDNA)

The significant role of cell-free DNA (cfDNA) for disease diagnosis, including cancer, has garnered a lot of attention. The challenges of creating target-specific primers and the possibility of false-positive signals make amplification-based detection methods problematic. Fluorescent biosensors based on CRISPR-Cas have been widely established, however they still require an amplification step before they can be used for detection. To detect cfDNA, researchers have created a CRISPR-Cas12a-based nucleic acid amplification-free fluorescent biosensor that uses a combination of fluorescence and colorimetric signaling improved by duplex-specific nuclease (DSN). DSN-assisted signal recycling is initiated in H1@MBs when the target cfDNA activates the CRISPR-Cas12a complex, leading to the degradation of single-strand DNA (ssDNA) sequences. This method has an extremely high detection limit for the BRCA-1 breast cancer gene. In addition to measuring viral DNA in a field-deployable and point-of-care testing (POCT) platform, this fast and highly selective sensor can be used to evaluate additional nucleic acid biomarkers.

Computer aided aptamer selection for fabrication of electrochemical sensor to detect Aflatoxin B1

Aflatoxin B1 (AFB1) is a naturally occurring toxin produced by Aspergillus flavus and Aspergillus parasiticus. The AFB1 is classified as a potent carcinogen and poses significant health risks both to humans and animals. Early detection of the toxin in post-harvest agricultural products will save lives and promote healthy food production. In this study, stratified docking approach was utilized to screen and identify potential aptamers that can bind to AFB1. ssDNA sequences were acquired from the Mendeley dataset, secondary and tertiary structures were predicted through a series of bioinformatics pipelines. Further, the final DNA tertiary structures were minimized and SiteMap algorithm was used to probe and locate binding cavities. According to the final XP docking result, a 34 nt sequence (5′-ATCCTGTGAGGAATGCTCATGCATAGCAAGGGCT-3′) aptamer with a docking score of −5.959 kcal/mol was considered for 200 ns MD Simulation. Finally, the screened DNA-aptamer was immobilized over the gold surface based on Au-S chemistry and utilized for the detection of AFB1. The fabricated DNA-aptamer electrode demonstrated a good analytical performance including wide linear range (1.0 to 1000 ng L−1), detection limit (1.0 ng L−1), high stability, and reproducibility. Communicated by Ramaswamy H. Sarma

Rapid and quantitative detection of DNA hybridization using a simplified Fabry-Perot interferometric biosensor.

This study introduces a miniaturized fiber-optic Fabry-Perot (FP) interferometric biosensor, distinctively engineered for cost-effective, rapid, and quantitative DNA sequence detection. By leveraging the interference patterns generated within a Fabry-Perot microcavity, our sensor precisely monitors DNA hybridization events in real-time. We have verified the sensor's biofunctionalization via fluorescent labeling and have extensively validated its performance through numerous hybridization and regeneration cycles with 1 μM single-stranded DNA (ssDNA) solutions. Demonstrating remarkable repeatability and reusability, the sensor effectively discerns ssDNA sequences exhibiting varying degrees of mismatches. Its ability to accurately distinguish between sequences with 2 and 7 mismatches underscores its potential as a valuable asset for swift DNA analysis. Characterized by its rapid response time-typically yielding results within 6 minutes-and its adeptness at mismatch identification, our biosensor stands as a potent tool for facilitating accelerated DNA diagnostics and research.

Mapping the Morphology of DNA on Carbon Nanotubes in Solution Using X-ray Scattering Interferometry.

Single-walled carbon nanotubes (SWCNTs) with adsorbed single-stranded DNA (ssDNA) are applied as sensors to investigate biological systems, with potential applications ranging from clinical diagnostics to agricultural biotechnology. Unique ssDNA sequences render SWCNTs selectively responsive to target analytes such as (GT)n-SWCNTs recognizing the neuromodulator, dopamine. It remains unclear how the ssDNA conformation on the SWCNT surface contributes to functionality, as observations have been limited to computational models or experiments under dehydrated conditions that differ substantially from the aqueous biological environments in which the nanosensors are applied. We demonstrate a direct mode of measuring in-solution ssDNA geometries on SWCNTs via X-ray scattering interferometry (XSI), which leverages the interference pattern produced by AuNP tags conjugated to ssDNA on the SWCNT surface. We employ XSI to quantify distinct surface-adsorbed morphologies for two (GT)n ssDNA oligomer lengths (n = 6, 15) that are used on SWCNTs in the context of dopamine sensing and measure the ssDNA conformational changes as a function of ionic strength and during dopamine interaction. We show that the shorter oligomer, (GT)6, adopts a more periodically ordered ring structure along the SWCNT axis (inter-ssDNA distance of 8.6 ± 0.3 nm), compared to the longer (GT)15 oligomer (most probable 5'-to-5' distance of 14.3 ± 1.1 nm). During molecular recognition, XSI reveals that dopamine elicits simultaneous axial elongation and radial constriction of adsorbed ssDNA on the SWCNT surface. Our approach using XSI to probe solution-phase morphologies of polymer-functionalized SWCNTs can be applied to yield insights into sensing mechanisms and inform future design strategies for nanoparticle-based sensors.

(Invited) Directed Evolution and Machine Learning for Improved ssDNA-SWCNTs Sensor for Nitric Oxide Detection

Single-walled carbon nanotubes (SWCNTs) show great potential for biosensing applications due to their unique optical properties. SWCNTs emit fluorescence in the near-infrared (NIR) region that is stable, biotransparent, and highly sensitive to changes in their environment. The biocompatibility and the selectivity of their interactions can be engineered through SWCNT surface functionalization. One of the most common functionalization approaches is the non-covalent wrapping of SWCNTs with single-stranded DNA (ssDNA). Though the ssDNA sequences can be varied to control the optical response of these SWCNT sensors, the dependence of the optical response on the sequence is unknown and unpredictable. This lack of information on the relationship between the ssDNA sequence and sensor performance is a major bottleneck in engineering SWCNT-based optical sensors.In this work, we develop a guided approach to engineer optical ssDNA-SWCNT sensors. Using a combination of machine learning and directed evolution[1], we designed an optical sensor for nitric oxide (NO), a pro-inflammatory mediator that induces inflammation[2]. We demonstrate a 7-fold enhancement in sensor response compared to the state-of-the-art sensor based on the (AT)15 sequence[3]. This approach thus provides a powerful means of overcoming existing bottlenecks in SWCNT sensor design, ushering a new generation of near-infrared technologies for biomedical research and clinical diagnostics.

Evolution of High-Affinity ssDNA Sequence to Carbon Nanotube

Colloidal ssDNA-wrapped single walled carbon nanotube (ssDNA-SWCNT) has been used as the second near-infrared (NIR-II, 1000 -1700 nm) fluorescent nanoprobes with outstanding biocompatibility, and acts as various optical nano sensors for biological analytes such as a dopamine and serotonin. The stability and affinity of ssDNA on the SWCNT surface significantly depends on the sequence of ssDNA, however the large-scale quantitative analysis between ssDNA sequence library, which has high affinity to the SWCNT surface, was repeatedly selected from a random ssDNA(N30) library (~1018) and the resulting sequence data was analyzed. The binding affinity of ssDNA to SWCNT was quantitatively characterized. To quantitatively analyze the kinetics of the process which occurs solvatochromic shift by sodium cholate, assumed the dynamics follow a first-order kinetic model and fit the experimental data using exponential regression, from which decay time constants were determined. As a result, the highest binding affinity of ssDNA to SWCNT was quantitatively characterized by time constant (1.5 X106 sec) which increased by 100 times compared to the N30 random library. Through analysis of the ssDNA sequences with high affinity for the SWCNT surface, we found that the sequences contain high contents of cytosine and adenine nucleotides and complementary base pairs appeared at distance of 5 to 8 bases. This novel sequence library can be applied for the preparation of the ultra-stable ssDNA-SWCNT dispersion and used for anchoring surface ligands for SWCNT as biosensor. Figure 1

What DNA Sequence Strongly Binds to Carbon Nanotube? High-Throughput Study

Noncovalent functionalization of single-walled carbon nanotubes (SWCNT) with amphiphilic polymers has significantly advanced the colloidal SWCNT efficacy for biomedical optical sensors. Single-strand DNA (ssDNA), as an amphiphilic biocompatible polymer, has been employed to fabricate ssDNA-wrapped SWCNT complexes, however the correlation between DNA sequence and wrapping affinity is not fully studied. In this research, we evolved and screened high-affinity ssDNA sequences from ~1015 ssDNA (random 30-mer), and analyzed the influence of DNA sequence on the wrapping affinity. Wrapping affinity of specific ssDNA sequence was quantitatively measured by the rate constant when ssDNA at the surface was exchanged to sodium cholate. The wrapping affinity relies on the base composition and k-mer motif. Also, we build a machine learning model to identify the high affinity DNA sequence with >85% accuracy. These results indicate the presence of “sequence motif(s)” to control the wrapping affinity and chemical conformation of ssDNA molecule on the SWCNT surface, which can be useful for developing DNA-assisted carbon nanosensors.

(Invited) Kinetics of SWCNT Coating Displacement By ssDNA Depends on SWCNT Structure

Non-covalent hybrids formed by suspending single-wall carbon nanotubes (SWCNTs) in single-stranded DNA (ssDNA) present novel phenomena and suggest potential applications in biomedicine and nanotube sorting. Although the structures of these hybrids have been simulated using molecular dynamics calculations, much more experimental research is needed to understand their properties. In this study, selective interactions between SWCNTs and ssDNA are probed by measuring the kinetics by which ssDNA displaces SDS (sodium dodecyl sulfate) coatings on the nanotube surfaces. We used samples initially suspended in a low concentration of SDS to allow the coating displacement to occur and to suppress formation of free SDS micelles. The displacement process was monitored by time-dependent measurements of SWCNT fluorescence intensities and wavelengths. Our experiments showed very smooth decreases in fluorescence intensity and red-shifts in fluorescence wavelengths during the displacement process. Through a series of measurements, we found that ssDNA sequences with repeated guanine (G) and thymine (T) bases showed systematic patterns in the coating displacement kinetics. One result is that SDS displacement depends strongly on oligo length, with (GT)3 showing an initial rate constant 500 times greater than that of (GT)20. For shorter oligos in the (GT) n series, we observe an inverse dependence of initial rate constant on SWCNT diameter, with SDS displacement from (6,5) more than twice as fast as from (8,7). However, this diameter dependence is not present for (GT) n oligos with n greater than 6. We also find a distinct and systematic dependence of initial rate constant on nanotube chiral angle for (GT)5 and (GT)6. This effect gives a factor of ~3 difference between (9,1) and (6,5) despite their identical diameters. We empirically find these initial rate constants to vary as (SWCNT diameter)-4(cos 3θ)-y with y = -1/2 for (GT)5 and -1 for (GT)6. In addition, the full displacement kinetic traces are well fitted by double exponential kinetics, with rate constants and amplitudes dependent on SWCNT diameter. A two-step kinetic model for the displacement process will be presented to account for these findings.

Molecular dynamics simulation of ssDNA and cationic polythiophene

In this work, molecular dynamics simulations of complexes composed of single strand DNA (ssDNA) sequences and cationic oligothiophenes are performed to understand experimental findings and the sensing ability of polythiophene electrolytes toward ssDNA. The simulation results exhibit no significant structural effect for replacing the cationic amine moiety with imidazole derivative on the side group of the oligomer. Adding a homopurine strand elongates the oligomer backbone; on the contrary, mixing up the homopyrimidine strand causes compression. On the other hand, these ssDNAs do not notably affect the compactness of the oligomer backbones. The anion-cation interactions play an essential role in the structural and spectroscopic change of cationic polythiophenes (CPTs) upon complexation with ssDNAs. The red shift of CPTs in the UV-VIS spectra with the addition of homopurine strands might be explained by the strong anion-cation, weak π −cation interactions, and high binding affinities. Nonpolar interactions (vdW and SA) and complex solvation energies dominate binding free energies. Hydrogen interaction analyses show that oligomers most likely approach the ssDNAs from their backbone upon complexation except for the duplex containing homopyrimidine strand and oligothiophene possessing imidazole derivative side chain.

Molecular test for COVID-19 diagnosis based on a colorimetric genomagnetic assay

The world is in a long pandemic period caused by the SARS-CoV-2 virus and massive diagnostic tests to assist efforts to control the spread of the disease and also to avoid new coronavirus variants are still needed. Herein, we propose a simple and accurate saliva-based colorimetric test for the diagnosis of COVID-19. Magnetic beads (MBs) modified with a sequence of single-strand DNA (ssDNA) complementary to the N gene of the SARS-CoV-2 RNA were developed and used for magnetic capture and separation from a complex saliva sample. A second biotinylated ssDNA sequence was applied, and the colorimetric detection was carried out by adding streptavidin-horseradish peroxidase conjugate, H2O2, and tetramethylbenzidine (TMB) as chromogenic substrate. The test does not require viral RNA isolation, transcription, or amplification steps and can be performed at room temperature. The molecular assay test can be run using 96-well microplates, allowing the diagnosis of a large number of samples in 90 min. A simple support for magnets was designed and constructed using a 3D printer that allows the magnetic separations directly in the 96-well microplate. The colorimetric test showed an excellent ability to discriminate between healthy individuals and patients infected with SARS-CoV-2, with 92% and 100% of clinical sensitivity and specificity, respectively. This performance was similar to that achieved using the gold standard RT-PCR technique. The proposed genomagnetic assay offers an opportunity to greatly increase population testing, contribute to controlling the spread of the virus, and improve health equity in testing for COVID-19.

Computation-Assisted Design of ssDNA Framework Nanorobots for Cancer Logical Recognition, Toehold Disintegration, Visual Dual-Diagnosis, and Synergistic Therapy.

Single-stranded DNA (ssDNA) allows flexible and directional modifications for multiple biological applications, while being greatly limited by their poor stability, increased folding errors, and complicated sequence optimizations. This greatly challenges the design and optimization of ssDNA sequences to fold stable 3D structures for diversified bioapplications. Herein, the stable pentahedral ssDNA framework nanorobots (ssDNA nanorobots) were intelligently designed, assisted by examining dynamic folding of ssDNA in self-assemblies via all-atom molecular dynamics simulations. Assisted by two functional siRNAs (S1 and S2), two ssDNA strands were successfully assembled into ssDNA nanorobots, which include five functional modules (skeleton fixation, logical dual recognition of tumor cell membrane proteins, enzyme loading, dual-miRNA detection and synergy siRNA loading) for multiple applications. By both theoretical calculations and experiments, ssDNA nanorobots were demonstrated to be stable, flexible, highly utilized with low folding errors. Thereafter, ssDNA nanorobots were successfully applied to logical dual-recognition targeting, efficient and cancer-selective internalization, visual dual-detection of miRNAs, selective siRNA delivery and synergistic gene silencing. This work has provided a computational pathway for constructing flexible and multifunctional ssDNA frameworks, enlarging biological application of nucleic acid nanostructures.

Primer Exchange Reaction Coupled with DNA-Templated Silver Nanoclusters for Label-Free and Sensitive Detection of MicroRNA.

MicroRNAs (MiRNAs) play pivotal roles in regulating gene expression, and serve as crucial biomarkers for diagnosis of a variety of disease. However, label-free and sensitive miRNA detection remains a huge challenge due to the low abundance. Herein, we developed an approach through integrating primer exchange reaction (PER) with DNA-templated silver nanoclusters (AgNCs) for label-free and sensitive miRNA detection. In this method, PER was used to amplify miRNA signals and produce single-strand DNA (ssDNA) sequences. The produced ssDNA sequences mediated DNA-templated AgNCs based signal generation by unfolding the designed hairpin probe (HP). The generated AgNCs signal was correlated with the dosage of target miRNA. Eventually, the established approach exhibited a low detection of limit of 47 fM with a great dynamic range of more than five orders of magnitude. In addition, the method was also utilized to detect the miRNA-31 expression in collected clinical samples from pancreatitis patients and demonstrated that miRNA-31 was upregulated in patients, showing a great promising of the method in clinical application.

Quantitative analyses of interactions between SpoVG and RNA/DNA

The Borrelia burgdorferi SpoVG protein has previously been found to be a DNA- and RNA-binding protein. To aid in the elucidation of ligand motifs, affinities for numerous RNAs, ssDNAs, and dsDNAs were measured and compared. The loci used in the study were spoVG, glpFKD, erpAB, bb0242, flaB, and ospAB, with particular focus on the untranslated 5′ portion of the mRNAs. Performing binding and competition assays yielded that the 5′ end of spoVG mRNA had the highest affinity while the lowest observed affinity was to the 5’ end of flaB mRNA. Mutagenesis studies of spoVG RNA and ssDNA sequences suggested that the formation of SpoVG-nucleic acid complexes are not entirely dependent on either sequence or structure. Additionally, exchanging uracil for thymine in ssDNAs did not affect protein-nucleic acid complex formation.

Design of a novel aptamer/molecularly imprinted polymer hybrid modified Ag–Au@Insulin nanoclusters/Au-gate-based MoS2 nanosheet field-effect transistor for attomolar detection of BRCA1 gene

Early detection of breast cancer, the first main cause of death in women, with robust assay platforms using appropriate biomarkers is of great importance for diagnosis and follow-up of the disease progression. This paper introduces an extra selective and sensitive label-free aptasensor for the screening of BRCA1 gene biomarker by taking advantage of a gate modified with aptamer and molecularly imprinted polymer hybrid (MIP) as a new synthetic receptor film coupled with an electrolyte-gated molybdenum disulfide (MoS2) field-effect transistor (FET). The Au gate surface of FET was modified with insulin stabilized bimetallic Ag–Au@nanoclusters (Ag–Au@InsNCs), after which, the immobilization of the hybridized aptamer and o-phenylenediamine was electropolymerized to form an aptamer-MIP hybrid receptor. The output characteristics of Apta-MIP hybrid modified Au gate MoS2 FET device were followed as a result of change in electrical double layer capacitance of electrolye-gate interface. The magnitude of decrease in the drain current showed a linear response over a wide concentration range of 10 aM to 1 nM of BRCA1 ssDNA with a sensitivity as high as 0.4851 μA/decade of concentration and a limit of detection (LOD) of 3.0 aM while very low responses observed for non-imprinted polymer. The devised aptasensor not only was capable to the discrimination of the complementary versus one-base mismatch BRCA1 ssDNA sequence, but also it could detect the complementary BRCA1 ssDNA in spiked human serum samples over a wide concentration range of 10 aM to 1.0 nM with a low LOD of 6.4 aM and a high sensitivity 0.3718 μA/decade.

Unraveling the molecular basis of protein search on ssDNA track and binding specificity of the ssDNA sequences.

The genetic materials of cellular organisms are organized as double-stranded DNA (dsDNA), which transiently gets unwrapped and forms single-stranded DNA (ssDNA) intermediates. These intermediates serve as a template for the various cellular processes. In its isolated form, ssDNA is vulnerable to nucleolytic degradation and prone to adopt unwanted secondary structures. To avoid the mess, cells employ specialized ssDNA binding proteins (SSBs), which bind with high affinity and both specifically and non-specifically to ssDNA to stabilize these intermediates. To understand their biophysics, we have developed a computational model that has been extensively parametrized by studying ssDNA binding on replication protein A, a major eukaryotic SSB, which is later tested on numerous other protein-ssDNA complexes. Our study delineates the molecular mechanism of target search by SSBs on the ssDNA track, which is otherwise difficult to capture through experimental approaches due to their binding promiscuity. The SSB-ssDNA model is then used as a testbed to develop a hybrid simulation technique for calculating protein-ssDNA binding thermodynamics for many ssDNA sequences in a single run. The method explores both sequence specificity of ssDNA and conformational space simultaneously by simulating a joint probability distribution, which makes searching through ssDNA sequence space computationally efficient. Our results show that the method agrees well with the experiments in predicting the relative binding specificity of ssDNA sequences for a given SSB without performing a costly, time-consuming DNA microarray experiment.

KAS-Analyzer: a novel computational framework for exploring KAS-seq data.

Kethoxal-assisted ssDNA sequencing (KAS-seq) is rapidly gaining popularity as a robust and effective approach to study the nascent dynamics of transcriptionally engaged RNA polymerases through profiling of genome-wide single-stranded DNA (ssDNA). Its latest variant, spKAS-seq, a strand-specific version of KAS-seq, has been developed to map genome-wide R-loop structures by detecting imbalances of ssDNA on two strands. However, user-friendly, open-source computational tools tailored for KAS-seq data are still lacking. Here, we introduce KAS-Analyzer, the first comprehensive computational framework aimed at streamlining and enhancing the analysis and interpretation of KAS-seq and spKAS-seq data. In addition to standard analyses, KAS-Analyzer offers many novel tools specifically designed for KAS-seq data, including, but not limited to: calculation of transcription-related metrics, identification of single-stranded transcribing (SST) enhancers, high-resolution mapping of R-loops, and differential RNA polymerase activity analysis. We provided a detailed overview of KAS-seq data and its diverse applications through the implementation of KAS-Analyzer. Using the example time-course KAS-seq datasets, we further showcase the robust capabilities of KAS-Analyzer for investigating dynamic transcriptional regulatory programs in response to UVB radiation. KAS-Analyzer is available at https://github.com/Ruitulyu/KAS-Analyzer.

Architecture of dual emissive three-dimensional nanostructure composites containing graphitic 2D sheets and iron oxide nanoparticles: detection of short single-stranded DNA sequences.

Here, we report an effect driven by repetitive heating and cooling; as a result, 2D and 1D nanomaterials are parallelly produced locally in a single reactor from the same precursors. Afterward, more repetitive heating and cooling induced the self-folding approach of a 2D nanomaterial with a 1D nanomaterial, giving them a self-assembled biconcave disk-shaped 3D nanostructure. The microscopy and spectroscopy studies reveal that the nanostructure has a diameter of nearly 200 nm and is composed of Fe, C, O and incorporated N and P. This 3D nanostructure composite shows red-shifted dual emission (430 nm and 500 nm) at two different excitations (350 nm and 450 nm), accompanied by a rare large Stokes shift (LSS), and it was employed in the detection of targeted short single-stranded DNA sequences (ssDNA). Upon the addition of target DNA, the specific binding of 3D nanostructure probes with the target triggers variations (off/on) of two signals, and by considering the decreased emission (fluorescence quenching) at 500 nm, we can detect the target ssDNA at the single-molecule level. The change of fluorescence intensity and the concentration of complementary target ssDNA sequences show a better linear relationship than a single emission-based probe, and the limit of detection (LOD) was as low as 0.47 nM.

An aptamer triple helix molecular switch for sensitive electrochemical assay of lipocalin 1 biomarker via dual signal amplifications.

Sensitively monitoring the concentration change of lipocalin 1 (LCN1) can provide data support for accurately diagnosing diabetic retinopathy and efficacy of treatment. Using a new aptamer triplex switch (ATS) probe and catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) dual signal amplifications, a highly sensitive electrochemical and enzyme-free biosensor for detecting LCN1 is reported. The ATS probes recognize and bind LCN1 to change its structure and release ssDNA sequences, which trigger the capture of methylene blue (MB)-tagged DNA on the sensor electrode via the CHA and HCR reactions. These MB tags are then subjected to electrochemical measurements to obtain highly amplified current outputs to sensitively detect LCN1 from 5 pM to 1 nM with a detection limit of 0.85 pM. This sensor also exhibits high interfering protein discrimination capability and can be employed to monitor low levels of LCN1 in diluted tear samples. Our established sensor significantly outperforms current major LCN1 detection methods based on immunoassays and thus holds promising potential for evaluating the severity of diabetic retinopathy and monitoring treatment response.