Homogeneous electrochemical ratiometric biosensor for MircoRNA detection based on UiO-66-NH2 signal probe and waste-free entropy-driven DNA machine.

The construction of efficient methods for highly sensitive and rapid detection of disease markers is essential for the early diagnosis of serious diseases. In this paper, taking advantage of the UiO-66-NH2 signal molecule in combination with a waste-free entropy-driven DNA machine, a novel homogeneous electrochemical ratiometric platform is developed to detect MircoRNA (miRNA). Metal-organic framework materials (UiO-66-NH2 MOF) and ferrocene were utilized as electrochemical signal tags and reference probes, respectively. The target-initiated waste-free three-dimensional (3D) entropy-driven DNA nanomachine is activated in the presence of miRNA, resulting in DNA-labeled-UiO-66-NH2 falling off from the electrode, leading to a decrease in the signal of UiO-66-NH2 at 0.83V. Our strategy can mitigate false positive responses induced by the DNA probes immobilized on electrodes in traditional distance-dependent signal adjustment ratiometric strategies. The proposed ratiometric platform demonstrates superior sensitivity (a detection limit of 9.8 fM), simplified operation, high selectivity, and high repeatability. The ratiometric biosensor is also applied to detect miRNA content in spiked serum samples.

Spherical Nucleic Acid-Mediated Spatial Matching-Guided Nonenzymatic DNA Circuits for the Prediction and Prevention of Malignant Tumor Invasion.

Detection of intracellular miRNAs, especially sensitive imaging of in vivo miRNAs, is vital to the precise prediction and timely prevention of tumorgenesis but remains a technical challenge in terms of nuclease resistance and signal amplification. Here, we demonstrate a gold nanoparticle-based spherical nucleic acid-mediated spatial matching-guided nonenzymatic DNA circuit (SSDC) for efficient screening of intracellular miRNAs and, in turn, finding cancerous tissues in living organisms before the appearance of clinical symptoms. Due to the substantially enhanced nuclease resistance, the false positive signal is avoided even in a complex biological medium. Target miRNA can straighten out the hairpin DNA probe to be linear, allowing the probe to penetrate into the internal region of a core/shell DNA-functionalized signal nanoampfilier and initiate a strand displacement reaction, generating an amplified fluorescence signal. The detection limit is as low as 17 pM, and miRNA imaging is in good accordance with the gold standard polymerase chain reaction method. The ability to image intracellular miRNAs is substantially superior to that of conventional fluorescence in situ hybridization techniques, making in vivo SSDC-based imaging competent for the precise prediction of tumorigenesis. By intratumoral chemotherapy guided by SSDC-based imaging, tumorigenesis and progression are efficiently controlled before the onset of clinical symptoms.

Triblock PolyA-Mediated Protein Biosensor Based on a Size-Matching Proximity Hybridization Analysis.

The antibodies in the natural biological world utilize bivalency/multivalency to achieve a higher affinity for antigen capture. However, mimicking this mechanism on the electrochemical sensing interface and enhancing biological affinity through precise spatial arrangement of bivalent aptamer probes still pose a challenge. In this study, we have developed a novel self-assembly layer (SAM) incorporating triblock polyA DNA to enable accurate organization of the aptamer probes on the interface, constructing a "lock-and-key-like" proximity hybridization assay (PHA) biosensor. The polyA fragment acts as an anchoring block with a strong affinity for the gold surface. Importantly, it connects the two DNA probes, facilitating one-to-one spatial proximity and enabling a controllable surface arrangement. By precisely adjusting the length of the polyA fragment, we can tailor the distance between the probes to match the molecular dimensions of the target protein. This design effectively enhances the affinity of the aptamers. Notably, our biosensor demonstrates exceptional specificity and sensitivity in detecting PDGF-BB, as confirmed through successful validation using human serum samples. Overall, our biosensor presents a novel and versatile interface for proximity assays, offering a significantly improved surface arrangement and detection performance.

A cubic DNA nanocage probe for in situ analysis of miRNA-10b in tumor-derived extracellular vesicles.

A cubic DNA nanocage probe is able to enter EVs derived from MDA-MB-231 cells and react with miRNA-10b. The probe-loaded EVs were employed to monitor the process of entry of miRNA-10b into MCF-10A cells, allowing visualization of EV-mediated intercellular communication of miRNA-10b between the cancer cells.

Live-cell imaging of human apurinic/apyrimidinic endonuclease 1 in the nucleus and nucleolus using a chaperone@DNA probe.

Human apurinic/apyrimidinic endonuclease 1 (APE1) plays crucial roles in repairing DNA damage and regulating RNA in the nucleus. However, direct visualization of nuclear APE1 in live cells remains challenging. Here, we report a chaperone@DNA probe for live-cell imaging of APE1 in the nucleus and nucleolus in real time. The probe is based on an assembly of phenylboronic acid modified avidin and biotin-labeled DNA containing an abasic site (named PB-ACP), which cleverly protects DNA from being nonspecifically destroyed while enabling targeted delivery of the probe to the nucleus. The PB-ACP construct specifically detects APE1 due to the high binding affinity of APE1 for both avidin and the abasic site in DNA. It is easy to prepare, biocompatible and allowing for long-term observation of APE1 activity. This molecular tool offers a powerful means to investigate the behavior of APE1 in the nuclei of various types of live cells, particularly for the development of improved cancer therapies targeting this protein.

In vitro detection of circulating tumor cells using the nicking endonuclease-assisted lanthanide metal luminescence amplification strategy.

The in vitro detection of circulating tumor cells (CTCs) has been proven as a vital method for early diagnosis and evaluation of cancer metastasis, since the existence and number fluctuation of CTCs have shown close correlation with clinical outcomes. However, it remains difficult and technically challenging to realize accurate CTCs detection, due to the rarity of CTCs in the blood samples with complex components. Herein, we reported a CTCs in vitro detection strategy, utilizing a loop amplification strategy based on DNA tetrahedron and nicking endonuclease reaction, as well as the anti-background interference based on lanthanide metal luminescence strategy. In this work, a detection system (ATDN-MLLPs) composed of an aptamer-functionalized tetrahedral DNA nanostructure (ATDN) and magnetic lanthanide luminescent particles (MLLPs) was developed. ATDN targeted the tumor cells via aptamer-antigen recognition and extended three hybridizable target DNA segments from the apex of a DNA tetrahedron to pair with probe DNA on MLLPs. Then, the nicking endonuclease (Nt.BbvCI) recognized the formed double-strand DNA and nicked the probe DNA to release the target DNA for recycling, and the released TbNps served as a high signal-to-noise ratio fluorescence signal source for CTCs detection. With a detection limit of 5 cells/mL, CTCs were selectively screened throughout a linear response range of low orders of magnitude. In addition, the ATDN-MLLPs system was attempted to detect possible existence of CTCs in biological samples in vitro.

DNA Nanospheres Assisted Spatial Confinement Signal Amplification for MicroRNA Imaging in Live Cancer Cells.

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.

Plasmonic Gold Nanostar-Based Probes with Distance-Dependent Plasmon-Enhanced Fluorescence for Ultrasensitive DNA Methyltransferase Assay.

The ultrasensitive DNA methyltransferase (Dam MTase) assay is of high significance for biomedical research and clinical diagnosis because of its profound effect on gene regulation. However, detection sensitivity is still limited by shortcomings, including photobleaching and weak signal intensities of conventional fluorophores at low concentrations. Plasmonic nanostructures with ultrastrong electromagnetic fields and fluorescence enhancement capability that can overcome these intrinsic defects hold great potential for ultrasensitive bioanalysis. Herein, a silica-coated gold nanostars (Au NSTs@SiO2)-based plasmon-enhanced fluorescence (PEF) probe with 20 "hot spots" was developed for ultrasensitive detection of Dam MTase. Here, the Dam Mtase assay was achieved by detecting the byproduct PPi of the rolling circle amplification reaction. It is worth noting that, benefiting from the excellent fluorescence enhancement capability of Au NSTs originating from their 20 "hot spots", the detection limit of Dam Mtase was reduced by nearly 105 times. Moreover, the proposed Au NST-based PEF probe enabled versatile evaluation of Dam MTase inhibitors as well as endogenous Dam MTase detection in GW5100 and JM110 Escherichia coli cell lysates, demonstrating its potential in biomedical analysis.

A novel fluorescence-electrochemiluminescence dual-mode sensing platform for high-precision BRAF gene detection.

In this study, we innovatively synthesized bipyridine ruthenium cluster nanosheets (RuMOFNCs), a novel metal-organic framework material that exhibits both fluorescence and electrochemiluminescence. Gold nanoparticles (AuNPs) were anchored onto RuMOFNCs via bipyridine chelation, enhancing optical signals and creating sites for attaching biologically functional probes. We employed tetraferrocene-modified DNA probes, linked via gold-sulfur (Au-S) bonds, to construct a dual-mode fluorescence-electrochemiluminescence biosensor. This sensor, exploiting exonuclease III (Exo III)-mediated cyclic amplification, inhibits the electron transfer from RuMOFNC to tetraferrocene, resulting in amplified fluorescence and electrochemiluminescence signals. The sensor demonstrates exceptional sensitivity for detecting the BRAF gene, with fluorescence and electrochemiluminescence detection limits of 10.3 aM (range: 0.1 fM to 1 nM) and 3.1 aM (range: 1 aM to 10 pM), respectively. These capabilities are attributed to RuMOFNCs' luminescence properties, tetraferrocene's quenching effect, and the specificity of base pairing. This study's findings hold substantial promise for biomedical research and clinical diagnostics, particularly in precision medicine and early disease detection.

Label-free and highly sensitive detection of microRNA from cancer cells via target-induced cascade amplification generation of lighting-up RNA aptamers.

The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.

Zipper-Confined DNA Nanoframe for High-Efficient and High-Contrast Imaging of Heterogeneous Tumor Cell.

Current study in the heterogeneity and physiological behavior of tumor cells is limited by the fluorescence in situ hybridization technology in terms of probe assembly efficiency, background suppression capability, and target compatibility. In a typically well-designed assay, hybridization probes are constructed in a confined nanostructure to achieve a rapid assembly for efficient signal response, while the excessively high local concentration between different probes inevitably leads to nonspecific background leakage. Inspired by the fabric zipper, we propose a novel confinement reaction pattern in a zipper-confined DNA nanoframe (ZCDN), where two kinds of hairpin probes are independently anchored respective tracks. The metastable states of the dual tracks can well avoid signal leakage caused by the nonspecific probe configuration change. Biomarker-mediated proximity ligation reduces the local distance of dual tracks, kinetically triggering an efficient allosteric chain reaction between the hairpin probes. This method circumvents nonspecific background leakage while maintaining a high efficiency in responding to targets. ZCDN is employed to track different cancer biomarkers located in both the cytoplasm and cytomembrane, of which the expression level and oligomerization behavior can provide crucial information regarding intratumoral heterogeneity. ZCDN exhibits high target response efficiency and strong background suppression capabilities and is compatible with various types of biological targets, thus providing a desirable tool for advanced molecular diagnostics.

DNA nanotechnology for nucleic acid analysis: sensing of nucleic acids with DNA junction-probes.

DNA nanotechnology deals with the design of non-naturally occurring DNA nanostructures that can be used in biotechnology, medicine, and diagnostics. In this study, we introduced a nucleic acid five-way junction (5WJ) structure for direct electrochemical analysis of full-length biological RNAs. To the best of our knowledge, this is the first report on the interrogation of such long nucleic acid sequences by hybridization probes attached to a solid support. A hairpin-shaped electrode-bound oligonucleotide hybridizes with three adaptor strands, one of which is labeled with methylene blue (MB). The four strands are combined into a 5WJ structure only in the presence of specific DNA or RNA analytes. Upon interrogation of a full-size 16S rRNA in the total RNA sample, the electrode-bound MB-labeled 5WJ association produces a higher signal-to-noise ratio than electrochemical nucleic acid biosensors of alternative design. This advantage was attributed to the favorable geometry on the 5WJ nanostructure formed on the electrode's surface. The 5WJ biosensor is a cost-efficient alternative to the traditional electrochemical biosensors for the analysis of nucleic acids due to the universal nature of both the electrode-bound and MB-labeled DNA components.

Correlated Hybrid DNA Structures Explored by the oxDNA Model.

Thermodynamically, perfect DNA hybridization can be formed between probes and their corresponding targets due to the favorable energy. However, this is not the case dynamically. Here, we use molecular dynamics (MD) simulations based on the oxDNA model to investigate the process of DNA microarray hybridization. In general, correlated hybrid DNA structures are formed, including one probe associated with several targets as well as one target hybrid with multiple probes leading to the target-mediated hybridization. The formation of these two types of correlated structures largely depends on the surface coverage of the DNA microarray. Moreover, DNA sequence, DNA length, and spacer length have an impact on the structural formation. Our findings shed light on the dynamics of DNA hybridization, which is important for the application of DNA microarray.

A fuel-initiated DNA molecular machine for microRNA detection in serum via poly-adenine-mediated spherical nucleic acids.

MicroRNAs (miRNAs) have been identified as promising disease diagnostic biomarkers. However, it is challenging to sensitively detect miRNAs, especially in complex biological environments, due to their low abundance and small size. Herein, we have developed a DNA-fueled molecular machine for sensitive detection of miRNA-22 (miR-22) in undiluted serum by combining poly-adenine-mediated spherical nucleic acids (polyA-SNAs) with a toehold mediated strand displacement reaction (TMSDR). The polyA-SNAs are constructed by the assembly of diblock DNA probes on a AuNP surface through the high binding affinity of polyA to AuNPs. The surface density of the diblock DNA probe can be controlled by tuning the length of the polyA block, and the orientation of the diblock DNA probe can adopt an upright conformation, which is beneficial to target hybridization and TMSDRs. TMSDR is an enzyme-free target recycling amplification approach. Taking advantage of polyA-mediated SNAs and TMSDR, the operation of the molecular machine based on two successive TMSDRs on polyA20-SNAs is rapid and efficient, which can significantly amplify the fluorescence response for detection of miR-22 in an undiluted complex matrix. The developed sensor can detect as low as 10 pM of target miRNA/DNA in undiluted fetal bovine serum within 30 min. The synergetic effect of polyA-mediated SNAs and TMSDR presents a potential alternative tool for the detection of biomarkers in real biological samples.

Enhanced DNA Entropy-Driven Circuit by Locked Nucleic Acids and Simulation-Guided Localization.

Signal amplification methods based on DNA molecular interactions are promising tools for detecting various biomarkers in low abundance. The entropy-driven circuit (EDC), as an enzyme-free signal amplification method, has been used in detecting and imaging a variety of biomarkers. The localization strategy can effectively increase the local concentration of the DNA reaction modules to improve the signal amplification effect. However, the localization strategy may also amplify the leak reaction of the EDC, and effective signal amplification can be limited by the unclear structure-function relationship. Herein, we utilized locked nucleic acid (LNA) modification to enhance the stability of the localized entropy-driven circuit (LEDC), which suppressed a 94.6% leak signal. The coarse-grained model molecular simulation was used to guide the structure design of the LEDC, and the influence of critical factors such as the localized distance and spacer length was analyzed at the molecular level to obtain the best reaction performance. The sensitivities of miR-21 and miR-141 detected by a simulation-guided optimal LEDC probe were 17.45 and 65 pM, 1345 and 521 times higher than free-EDC, respectively. The LEDC was further employed for the fluorescence imaging of miRNA in cancer cells, showing excellent specificity and sensitivity. This work utilizes LNA and molecular simulations to comprehensively improve the performance of a localized DNA signal amplification circuit, providing an advanced DNA probe design strategy for biosensing and imaging as well as valuable information for the designers of DNA-based probes.

Electrochemically-gated graphene broadband microwave waveguides for ultrasensitive biosensing.

Identification of non-amplified DNA sequences and single-base mutations is essential for molecular biology and genetic diagnostics. This paper reports a novel sensor consisting of electrochemically-gated graphene coplanar waveguides coupled with a microfluidic channel. Upon exposure to analytes, propagation of electromagnetic waves in the waveguides is modified as a result of interactions with the fringing field and modulation of graphene dynamic conductivity resulting from electrostatic gating. Probe DNA sequences are immobilised on the graphene surface, and the sensor is exposed to DNA sequences which either perfectly match the probe, contain a single-base mismatch or are unrelated. By monitoring the scattering parameters at frequencies between 50 MHz and 50 GHz, unambiguous and reproducible discrimination of the different strands is achieved at concentrations as low as one attomole per litre (1 aM). By controlling and synchronising frequency sweeps, electrochemical gating, and liquid flow in the microfluidic channel, the sensor generates multidimensional datasets. Advanced data analysis techniques are utilised to take full advantage of the richness of the dataset. A classification accuracy >97% between all three sequences is achieved using different Machine Learning models, even in the presence of simulated noise and low signal-to-noise ratios. The sensor exceeds state-of-the-art sensitivity of field-effect transistors and microwave sensors for the identification of single-base mismatches.

Nanomechanoelectrical approach to highly sensitive and specific label-free DNA detection.

Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical methods, which solely rely on measuring electrical signals in transducers during probe-target DNA hybridization, are prone to nonspecific electrostatic and electrochemical interactions, subsequently limiting their specificity and detection limit. Here, we demonstrate a nanomechanoelectrical approach that delivers ultra-robust specificity and a 100-fold improvement in detection limit. We drive nanostructural DNA strands tethered to a graphene transistor to oscillate in an alternating electric field and show that the transistor-current spectra are characteristic and indicative of DNA hybridization. We find that the inherent difference in pliability between unpaired and paired DNA strands leads to the spectral characteristics with minimal influence from nonspecific electrostatic and electrochemical interactions, resulting in high selectivity and sensitivity. Our results highlight the potential of high-performance DNA analysis based on miniaturized all-electronic settings.

Lateral flow DNA biosensor for visual detection of nucleic acid with triple-helix DNA functionalized carbon nanotube.

We describe a novel lateral flow DNA biosensor (LFDB) based on carbon nanotube (CNT) and triple helix DNA (THD). The carboxylated CNT was first conjugated with amine-modified auxiliary single-stranded DNA probe (P1) by dehydration reaction and used as signal probe. A main DNA probe (P0) was introduced to react with the P1 and formed the THD on the CNT surface. Because of the large spatial effect, P1 was in an inactive state and cannot hybridize with the capture DNA probe (P2) fixed on the LFDB test area. When the target DNA was present, P0 in the triple helix DNA hybridized with the target DNA due to the stronger base action, and the decomposition of the triple helix structure exposed P1. Therefore, P1 on CNT surface was activated to hybridize with P2. The CNT along with P1 was thus captured at the test area and accumulated to show a black line, which can be observed by naked eye for qualitative analysis and recorded with a portable grayscale reader for quantitative analysis. Single-stranded DNA was used as a target to prove the feasibility of the model. Under the best experimental conditions, the THD-CNT based LFDB was able to detect the lowest DNA concentration of 15 pM, which is 2.67 times better than that of the traditional duplex CNT-based LFDB. It should be noted that the LFDB based on THD functionalized CNT can differentiate between one-base-mismatched DNA and the complementary target DNA, can detected target DNA in 10% human serum, and can be employed as a versatile platform to detect various target (proteins, small molecular) by changing the sequence of P0. This biosensor platform has enormous potential in the point-of-care detection of a rich diversity of analytes for clinical diagnosis and biomedical research.

All-Covalent Nuclease-Resistant and Hydrogel-Tethered DNA Hairpin Probes Map pN Cell Traction Forces.

Cells sense and respond to the physical properties of their environment through receptor-mediated signaling, a process known as mechanotransduction, which can modulate critical cellular functions such as proliferation, differentiation, and survival. At the molecular level, cell adhesion receptors, such as integrins, transmit piconewton (pN)-scale forces to the extracellular matrix, and the magnitude of the force plays a critical role in cell signaling. The most sensitive approach to measuring integrin forces involves DNA hairpin-based sensors, which are used to quantify and map forces in living cells. Despite the broad use of DNA hairpin sensors to study a variety of mechanotransduction processes, these sensors are typically anchored to rigid glass slides, which are orders of magnitude stiffer than the extracellular matrix and hence modulate native biological responses. Here, we have developed nuclease-resistant DNA hairpin probes that are all covalently tethered to PEG hydrogels to image cell traction forces on physiologically relevant substrate stiffness. Using HeLa cells as a model cell line, we show that the molecular forces transmitted by integrins are highly sensitive to the bulk modulus of the substrate, and cells cultured on the 6 and 13 kPa gels produced a greater number of hairpin unfolding events compared to the 2 kPa substrates. Tension signals are spatially colocalized with pY118-paxillin, confirming focal adhesion-mediated probe opening. Additionally, we found that integrin forces are greater than 5.8 pN but less than 19 pN on 13 kPa gels. This work provides a general strategy to integrate molecular tension probes into hydrogels, which can better mimic in vivo mechanotransduction.

Bio-barcode assay: A useful technology for ultrasensitive and logic-controlled specific detection in food safety: A review.

Food safety is one of the greatest public health challenges. Developing ultrasensitive detection methods for analytes at ultra-trace levels is, therefore, essential. In recent years, the bio-barcode assay (BCA) has emerged as an effective ultrasensitive detection strategy that is based on the indirect amplification of various DNA probes. This review systematically summarizes the progress of fluorescence, PCR, and colorimetry-based BCA methods for the detection of various contaminants, including pathogenic bacteria, toxins, pesticides, antibiotics, and other chemical substances in food in over 120 research papers. Current challenges, including long experimental times and strict storage conditions, and the prospects for the application of BCA in biomedicine and environmental analyses, have also been discussed herein.