Catalytic Hairpin Assembly Research Articles

Proximity-Guaranteed DNA Machine for Accurate Identification of Breast Cancer Extracellular Vesicles.

Breast cancer is one of the most diagnosed cancers worldwide. Precise diagnosis and subtyping have important significance for targeted therapy and prognosis prediction of breast cancer. Herein, we design a proximity-guaranteed DNA machine for accurate identification of breast cancer extracellular vesicles (EVs), which is beneficial to explore the subtype features of breast cancer. In our design, two proximity probes are located close on the same EV through specific recognition of coexisting surface biomarkers, thus being ligated with the help of click chemistry. Then, the ligated product initiates the operation of a DNA machine involving catalytic hairpin assembly and clusters of regularly interspaced short palindromic repeats (CRISPR)-Cas12a-mediated trans-cleavage, which finally generates a significant response that enables the identification of EVs expressing both biomarkers. Principle-of-proof studies are performed using EVs derived from the breast cancer cell line BT474 as the models, confirming the high sensitivity and specificity of the DNA machine. When further applied to clinical samples, the DNA machine is shown to be capable of not only distinguishing breast cancer patients with special subtypes but also realizing the tumor staging regarding the disease progression. Therefore, our work may provide new insights into the subtype-based diagnosis of breast cancer as well as identification of more potential therapeutic targets in the future.

Optical Nanobiosensor Based on Surface-Enhanced Raman Spectroscopy and Catalytic Hairpin Assembly for Early-Stage Lung Cancer Detection via Blood Circular RNA.

Lung cancer has become the leading cause of cancer-related deaths globally. However, early detection of lung cancer remains challenging, resulting in poor outcomes for the patients. Herein, we developed an optical biosensor integrating surface-enhanced Raman spectroscopy (SERS) with a catalyzed hairpin assembly (CHA) to detect circular RNA (circRNA) associated with tumor formation and progression (circSATB2). The signals of the Raman reporter were considerably enhanced by generating abundant SERS "hot spots" with a core-shell nanoprobe and 2D SERS substrate with calibration capabilities. This approach enabled the sensitive (limit of detection: 0.766 fM) and reliable quantitative detection of the target circRNA. Further, we used the developed biosensor to detect the circRNA in human serum samples, revealing that patients with lung cancer had higher circRNA concentrations than healthy subjects. Moreover, we characterized the unique circRNA concentration profiles of the early stages (IA and IB) and subtypes (IA1, IA2, and IA3) of lung cancer. These results demonstrate the potential of the proposed optical sensing nanoplatform as a liquid biopsy and prognostic tool for the early screening of lung cancer.

A dual-signal ratiometric electrochemical aptasensor based on Thi/Au/ZIF-8 and catalytic hairpin assembly for ultra-sensitive detection of aflatoxin B1.

Adual-signal ratiometric electrochemical aptasensor has been developed for AFB1 detection using thionine/Au/zeolitic imidazolate framework-8 (Thi/Au/ZIF-8) nanomaterials and catalytic hairpin assembly (CHA) reaction. Thi/Au/ZIF-8 combined with DNA hairpin 2 (H2) was used as a signal probe. [Fe(CN)6]3-/4- was served as another signal probe, and the IThi/Au/ZIF-8/I[Fe(CN)6]3-/4- ratio was for the first timeutilized to quantify AFB1. AFB1-induced CHA was used to expand the ratio of electrical signals. In the presence of AFB1, H2/Thi/Au/ZIF-8 bound to the electrode via CHA, enhanced the current signal of Thi/Au/ZIF-8. H2 contained the DNA phosphate backbone hindered [Fe(CN)6]3-/4- redox reaction and resulted in a lower [Fe(CN)6]3-/4- current signal. This aptasensor exhibited high specificity for AFB1, a linear range of 0.1pgmL-1 to 100ngmL-1, and a detection limit of 0.089pgmL-1. It demonstrated favorable sensitivity, selectivity, stability, and repeatability. The aptasensor was suitable for detecting AFB1 in peanuts and black tea and holds potential for real sample applications.

Surface confinement–induced electrochemiluminescence enhancement of Au nanoclusters for ultrasensitive detection of a soluble urokinase-type plasminogen activator receptor

In this study, gold nanoclusters (Au NCs) anchored on the surface of 2D Zn-containing hydroxyl double salt (Zn-HDS) with surface confinement–induced electrochemiluminescence (SC-ECL) improvement were used as an intensive ECL emitter to construct a biosensor for ultrasensitive detection of soluble urokinase-type plasminogen activator receptor (suPAR) related to cardiovascular disease in chronic nephritis patients. Impressively, SC-ECL of Au NCs/Zn-HDS nanocomposites exhibited fast electrochemical excitation and efficient radiation transition, showing 4.9 times stronger ECL emission and 10.9 times higher ECL efficiency than those of pure Au NCs. This improvement was attributed to the matched energy level of Zn-HDS and Au NCs, enabling electron–hole pairs of Au NCs to be trapped by the energy levels of Zn-HDS. Moreover, a bipedal DNA walker was generated through target-induced DNA strand displacement and catalytic hairpin assembly (CHA), which was attached to a stable DNA cubic track through electrostatic attraction of positively charged SH-PEG-NH3+ to improve walking efficiency and further boost detection sensitivity. Consequently, the proposed sensing platform achieved trace detection of suPAR ranging from 0.700 pg/mL to 80.0 ng/mL with a low detection limit of 0.344 pg/mL and was successfully applied to detect suPAR in patient serum. This work synthesized a highly efficient metal nanocluster-based ECL emitter, showing great potential in constructing ultrasensitive biosensors for early disease diagnosis.

An enzymatically activated AND-gate DNA logic circuit for tumor cells recognition via multi-microRNAs detection

The DNA-based logic circuit, constructed to mimic biochemical reaction networks, is highly significant in detecting biomarkers at the molecular level. The differences in the expression levels of microRNAs (miRNAs) within different types of cells provide hope for distinguishing cell subtypes. However, reliance on a single miRNA often leads to unreliable results. Herein, we constructed an enzyme-triggered cascade logic circuit based on the AND gate, which is capable of generating corresponding fluorescence signals in the presence of target miRNAs. The introduction of apurinic/apyrimidinic (AP) sites effectively reduces the likelihood of false signal generation. Amplification of the fluorescence signal relies on the catalytic hairpin assembly and the repetitive reuse of the multicomponent nucleic acid enzyme (MNAzyme). We demonstrated that the logic circuit can not only distinguish cancer cells from normal cells but also identify different types of cancer cells. The programmability of the logic circuits and the simplicity of the assay system allow us to modify the functional sequences to recognize different types of biomarkers, thus providing a reference for the identification of various cell subtypes.

A leakage-resistant catalytic hairpin assembly for ultrasensitive detection of miRNA-146a in systemic lupus erythematosus

a leakage-resistant catalytic hairpin assembly (CHA) was developed for ultrasensitive detection of miRNA-146a in systemic lupus erythematosus. miRNA-146a can trigger the hybridization chain reaction (HCR) and form a long duplex. The two side chain of duplex can cleave the leakage-resistant hairpin and induce the CHA reaction. Simultaneously, the produced DNA fragment can also circularly catalyze and fasten the HCR reaction. The leakage-resistant hairpin used for CHA was firstly locked by substrate strand DNA. The special design inhibits the circuit leakage between hairpins, causing lower background than those traditional hairpin design. The limit of detection was improved to 1 fM by HCR coupled leakage-resistant CHA strategy. Significantly, it possibly provided an alternative strategy to prevent the leakage between hairpins. In addition, the incubation time had been obviously shorted by the recycle using of cleaved DNA fragments. It also showed a great specificity for miRNA-146a towards other similar sequence and great potential in early screening and clinical diagnosis.

DNA Framework-Based Programmable Atom-Like Nanoparticles for Non-Coding RNA Recognition and Differentiation of Cancer Cells.

The cooperative diagnosis of non-coding RNAs (ncRNAs) can accurately reflect the state of cell differentiation and classification, laying the foundation of precision medicine. However, there are still challenges in simultaneous analyses of multiple ncRNAs and the integration of biomarker data for cell typing. In this study, DNA framework-based programmable atom-like nanoparticles (PANs) are designed to develop molecular classifiers for intra-cellular imaging of multiple ncRNAs associated with cell differentiation. The PANs-based molecular classifier facilitates signal amplification through the catalytic hairpin assembly. The interaction between PAN reporters and ncRNAs enables high-fidelity conversion of ncRNAs expression level into binding events, and the assessment of in situ ncRNAs levels via measurement of the fluorescent signal changes of PAN reporters. Compared to non-amplified methods, the detection limits of PANs are reduced by four orders of magnitude. Using human gastric cancer cell lines as a model system, the PANs-based molecular classifier demonstrates its capacity to measure multiple ncRNAs in living cells and assesses the degree of cell differentiation. This approach can serve as a universal strategy for the classification of cancer cells during malignant transformation and tumor progression.

CRISPR-Cas12a-activated proximity catalytic hairpin assembly strategy for sensitive gene analysis in Streptococcus mutans

The cnm gene in Streptococcus mutans (S. mutans) mediates bacterial binding to extracellular matrix and invasion within host cells, and has been found to be correlated with the occurrence of brain microbleeds. Hence, it is crucial to develop a reliable method to evaluate cnm-positive S. mutans. Herein, we propose a novel diagnostic platform utilizing Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for accurate identification of the cnm gene, along with proximity catalytic hairpin assembly (CHA) for enhanced signal amplification, for the detection and analysis of the cnm gene in S. mutans. When the cnm gene is present, it activates trans-cleavage activity of Cas12a to trigger the proximity CHA. In contrast to conventional CHA, the hairpin probes in proximity CHA were linked in a sequential manner using a liner ssDNA sequence. Additionally, the CHA process was facilitated by the Duplex-Specific Nuclease (DSN), which enhanced the efficiency and speed of the reaction. The approach exhibits several notable benefits, including i) the target recognition capability of crRNA in CRISPR-Cas system enhances the detection specificity; ii) Cas12a-crRNA is independent on the PAM during recognizing target dsDNA; iii) the use of the DSN and the proximity design of the H1-H2 complex has significantly enhanced the reactive efficiency of proximity CHA. As a result, the method exhibits a low limit of detection of 47 aM and a high selectivity to cnm gene. Moreover, the approach has been effectively employed in clinical settings to accurately differentiate S. mutans from a combination of microorganisms.

A triple-amplified electrochemical-photoelectrochemical dual-mode biosensing platform for viral gene assay based on catalytic hairpin assembly, CRISPR-Cas12a and liposome-entrapped bifunctional methylene blue

Taking SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene as the model due to the great destructiveness of COVID-19 in the past years, a triple-amplified electrochemical-photoelectrochemical (EC-PEC) dual-mode biosensing platform was constructed for viral gene assay based on catalytic hairpin assembly (CHA), CRISPR-Cas12a and liposome-entrapped bifunctional methylene blue. Firstly, the SARS-CoV-2 RdRp gene triggered CHA nonenzymatic amplification process to produce large amounts of double-stranded DNA (dsDNA) that could activate CRISPR-Cas12a trans-cleavage ability. Subsequently, the activated CRISPR-Cas12a trans-cleaved nearby ssDNA carrying methylene blue (MB)-encapsulated liposome sphere (MELS) and streptavidin-modified magnetic bead (SMB), and lots of MELS were released. After magnetic separation, MELS was lysed by Triton X-100, releasing a large quantity of bifunctional MB molecules that not only generate an obvious EC signal originated from the electro-oxidation of MB, but also effectively induce the photocurrent-polarity-switching of the Sb2S3 NRs-modified photoelectrode from cathodic photocurrent to anodic photocurrent and generate a large anodic photocurrent. Based on the combination of the above triple amplification processes with MB-induced photocurrent-polarity-switching and EC-PEC dual-mode sensing strategies, the SARS CoV-2 RdRp gene was sensitively, selectively and accurately assayed with a linear response range of 0.1 fM – 10nM (1 aM – 10nM) and a detection limit of 69.6 aM (0.11 aM) for EC (PEC) method. The proposed EC-PEC dual-mode biosensor is also easily extended to the analysis of other viral nucleic acid by changing the related sequences of H1 and H2 DNAs, providing a new way in the early diagnosis and epidemic control of diseases caused by viruses.

An enzyme-free sensing platform for miRNA detection and in situ imaging in clinical samples based on DNAzyme cleavage-triggered catalytic hairpin assembly

MicroRNA (miRNA) is demonstrated to be associated with the occurrence and development of various diseases including cancer. Currently, most miRNA detection methods are confined to in vitro detection and cannot obtain information on the temporal and spatial expression of miRNA in relevant tissues and cells. In this work, we established a novel enzyme-free method that can be applied to both in vitro detection and in situ imaging of miRNA by integrating DNAzyme and catalytic hairpin assembly (CHA) circuits. This developed CHA-Amplified DNAzyme miRNA (CHAzymi) detection system can realize the quantitively in vitro detection of miR-146b (the biomarker of papillary thyroid carcinoma, PTC) ranging from 25 fmol to 625 fmol. This strategy has also been successfully applied to in situ imaging of miR-146b both in human PTC cell TPC-1 and clinical samples, showing its capacity as an alternative diagnostic method for PTC. Furthermore, this CHAzymi system can be employed as a versatile sensing platform for various miRNAs by revising the relevant sequences. The results imply that this system may expand the modality of miRNA detection and show promise as a novel diagnostic tool in clinical settings, providing valuable insights for effective treatment and management of the disease.

A new strategy based on a cascade amplification strategy biosensor for on-site eDNA detection and outbreak warning of crown-of-thorns starfish

Population outbreaks of the crown-of-thorns starfish (COTS) seriously threaten the sustainability of coral reef ecosystems. However, traditional ecological monitoring techniques cannot provide early warning before the outbreaks, thus preventing timely intervention. Therefore, there is an urgent need for a more accurate and faster technology to predict the outbreaks of COTS. In this work, we developed an electrochemical biosensor based on a programmed catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cyclic amplification strategy for sensitive and selective detection of COTS environmental DNA (eDNA) in water bodies. This biosensor exhibited excellent electrochemical characteristics, including a low limit of detection (LOD = 18.4 fM), low limit of quantification (LOQ = 41.1 fM), and wide linear range (50 fM – 10 nM). The biosensing technology successfully allowed the detection of COTS eDNA in the aquarium environment, and the results also demonstrated a significant correlation between eDNA concentration and COTS number (r = 0.990; P < 0.001). The reliability and accuracy of the biosensor results have been further validated through comparison with digital droplet PCR (ddPCR). Moreover, the applicability and accuracy of the biosensor were reconfirmed in field tests at the COTS outbreak site in the South China Sea, which has shown potential application in dynamically monitoring the larvae before the COTS outbreak. Therefore, this efficient electrochemical biosensing technology offers a new solution for on-site monitoring and early warning of the COTS outbreak.

Self-assembly of hyperbranched DNA network structure for signal amplification detection of miRNA

Catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) can achieve the high sensitivity and rapid reaction rate in detecting miRNA. However, the amplification efficiency by these methods are limited. Herein, an enzyme-free and label-free hyperbranched DNA network structure (HDNS) was designed, in which localized catalytic hairpin assembly (LCHA) and hybridization chain reaction occurred in the horizontal axis and longitudinal axis, respectively, exhibiting intensive signal dual-amplification. miRNA-122 was selected as the target on behalf of miRNA to design the HDNS sensor. The fluorescence signal change of HDNS showed good linearity for detecting miRNA-122 in the concentration range from 0.1 nM to 60 nM with a limit of detection (LOD) at 37 pM which was lower than those of the sensors based on separate CHA or HCR. Afterwards, the HDNS sensor was applied to detect miRNA-122 in serum samples with the recovery rate in the range of 97.2 %–107 %. The sensor could distinguish different kinds of miRNAs, even the family members with high sequence homology, exhibiting excellent selectivity. This method provided a novel design strategy for improving the sensitivity and selectivity of DNA sensor for miRNA detection.

Dual-Encoded Affinity Microbead Signature Combinatorial Profiling for Acute Myocardial Infarction High-Sensitivity Diagnosis.

The early diagnosis of acute myocardial infarction (AMI) is dependent on the combined feedback of multiple cardiac biomarkers. However, it remains challenging to precisely detect multicardiac biomarkers in complex blood early due to the lack of sensitive and specific diagnostic indicators and the low abundance and small size of associated biomarkers with high specificity (such as microRNAs). To make matters worse, spectral overlap significantly limits the multiplex analysis of cardiac biomarkers by fluorescent probes, leading to bias in the diagnosis of myocardial infarction. Herein, we developed a method for simultaneous detection of miRNAs and protein biomarkers using size- and color-coded microbeads that carry signature for target capture. We also constructed a microfluidic chip with different spacer arrays that segregate these microbeads in different chip regions according to their size to produce signature signals, indicating the level of different biomarkers. The signals on the microbeads were hugely amplified by catalytic hairpin assembly and rolling circle amplification. Notably, this strategy enables the simultaneous and in situ sensitive profiling of six kinds of biomarkers via adding two different fluorescent labels, removing the limitations of spectral overlap. We envision that the strategy has great potential for application in clinical diagnosis for AMI.

The tightest self-assembled ruthenium metal-organic framework combined with proximity hybridization for ultrasensitive electrochemiluminescence analysis of paraquat.

Metal-organic frameworks (MOFs), as porous materials, have great potential for exploring high-performance electrochemiluminescence (ECL) probes. However, the constrained applicability of MOFs in the realm of ECL biosensing is primarily attributed to their inadequate water stability, which consequently impairs the overall ECL efficiency. Herein, we developed a competitive ECL biosensor based on a novel tightest structural ruthenium-based organic framework emitter combining the proximity hybridization-induced catalytic hairpin assembly (CHA) strategy and the quenching effect between the Ru-MOF and ferrocene for detecting paraquat (PQ). Through a simple hydrothermal synthesis strategy, ruthenium and 2,2'-bipyrimidine (bpm) are head-to-head self-assembled to obtain a novel tightest structural Ru-MOF. Due to the metal-ligand charge-transfer (MLCT) effect between ruthenium and the bpm ligand and the connectivity between the internal chromophore units, the Ru-MOF exhibits strong ECL emissions. Meanwhile, the coordination-driven Ru-MOF utilizes strong metal-organic coordination bonds as building blocks, which effectively solves the problem of serious leakage of chromophores caused by water solubility. The sensitive analysis of PQ is realized in the range of 1 pg/mL to 1 ng/mL with a detection limit of 0.352 pg/mL. The tightest structural Ru-MOF driven by the coordination of ruthenium and bridging ligands (2,2'-bipyrimidine, bpm) provides new horizons for exploring high-performance MOF-based ECL probes for quantitative analysis of biomarkers.

Visually evaluating drug efficacy in living cells using COF-based fluorescent nanoprobe via CHA amplified detection of miRNA and simultaneous apoptosis imaging

BackgroundsCancer is a highly fatal disease which is close relative of miRNA aberrant expression and apoptosis disorders. Elucidation of the therapeutic efficacy through investigating the changes in miRNA and apoptosis holds immense importance in advancing the development of miRNA-based precision therapy. However, it remains a challenge as how to visually evaluate the efficacy during protocol optimization of miRNA-based anticancer drugs at the cellular level. Therefore, exploring effective and noninvasive methods for real-time monitoring of therapeutic efficacy in living cells is of great significance. ResultsHerein, we reported a novel fluorescent nanoprobe COF–H1/H2-Peptide for visually evaluating drug efficacy in living cells through amplified imaging of low-abundant miRNA-221 with catalytic hairpin assembly (CHA) circle amplification, as well as simultaneous caspase-3 imaging. With strong stability and good biocompatibility, this newly fabricated amplified nanoprobe showed high sensitivity and specificity for the detection of miRNA-221 and caspase-3, and the limit of detection (LOD) of miRNA-221 was as low as 2.79 pM. The fluorescent imaging results showed that this amplified nanoprobe could not only detect caspase-3 in living cells, but also effectively detect low levels of miRNA-221 with increasing anticancer drug concentration and treatment time. The smart nanoprobe had effective performance for optimizing miRNA-based drug treatment schedules by dual-color fluorescence imaging. SignificanceThis nanoprobe combined CHA amplified detection of intracellular miRNA-221 and synchronous apoptosis imaging, with excellent sensitivity for the detection of cellular low-level miRNA, enabling the realization of real-time assessment of the efficacy of miRNA-based therapy in living cells. This work presents a promising approach for revealing the regulatory mechanisms between miRNAs and apoptosis in cancer occurrence, development, and treatment.

Label-free electrochemical sensing platform for sensitive detection of ampicillin by combining nucleic acid isothermal enzyme-free amplification circuits with CRISPR/Cas12a

The residue of ampicillin (AMP) in food and ecological environment poses a potential harm to human health. Therefore, a reliable system for detecting AMP is in great demand. Herein, a label-free and sensitive electrochemical sensor utilizing NH2–Co-MOF as an electrocatalytic active material for methylene blue (MB) was developed for rapid and facile AMP detection by combining hybridization chain reaction (HCR), catalytic hairpin assembly (CHA) with CRISPR/Cas12a. The surface of glassy carbon electrode modified with NH2–Co-MOF was able to undergo HCR independent of the AMP, forming long dsDNA complexes to load MB, resulting in strong original electrochemical signal. The presence of AMP could trigger upstream CHA circuit to activate the CRISPR/Cas12a system, thereby achieving rapid non-specific cleavage of the trigger ssDNA of HCR on the electrode surface, hindering the occurrence of HCR and reducing the load of MB. Significant signal change triggered by the target was ultimately obtained, thus achieving sensitive detection of the AMP with a detection limit as low as 1.60 pM (S/N = 3). The proposed sensor exhibited good stability, selectivity, and stability, and achieved reliable detection of AMP in milk and livestock wastewater samples, demonstrating its promising application prospects in food safety and environmental monitoring.

Nucleic Acid and Nanomaterial Synergistic Amplification Enables Dual Targets of Ultrasensitive Fluorescence Quantification to Improve the Efficacy of Clinical Tuberculosis Diagnosis.

Interferon-γ (IFN-γ) release assays (IGRAs) are constrained by the limited diagnostic performance of a single indicator and the excessive Mycobacterium tuberculosis (Mtb) antigen stimulation time. This study presents a simultaneous, homogeneous, rapid, and ultrasensitive fluorescence quantification strategy for IFN-γ and IFN-γ-induced protein 10 (IP-10). This method relies on the high-affinity binding of aptamers to IFN-γ and IP-10, the enzyme-free catalytic hairpin assembly reaction, and the heightened sensitivity of CdTe quantum dots to Ag+ and hairpin structure C-Ag+-C and carbon dots to Hg2+ and hairpin structure T-Hg2+-T. Under optimized conditions, the selectivity of IFN-γ and IP-10 was excellent, with a linear range spanning from 1 to 100 ag/mL and low limits of detection of 0.3 and 0.5 ag/mL, respectively. Clinical practicality was confirmed through testing of 57 clinical samples. The dual-indicator combination detection showed 92.8% specificity and 93.1% sensitivity, with an area under the curve of 0.899, representing an improvement over the single-indicator approach. The Mtb antigen stimulation time was reduced to 8 h for 6/7 clinical samples. These findings underscore the potential of our approach to enhance the efficiency and performance of a tuberculosis (TB) clinical diagnosis.

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.

Rapid and accurate SERS assay of disease-related nucleic acids based on isothermal cascade signal amplifications of CRISPR/Cas13a system and catalytic hairpin assembly

Developing rapid, accurate and convenient nucleic acid diagnostic techniques is essential for the prevention and control of contagious diseases that are prone to gene mutations and may have homologous sequences, especially emerging infectious diseases such as the SARS-CoV-2 pandemic. Herein, a one-pot SERS assay integrating isothermal cascade signal amplification strategy (i.e., CRISPR/Cas13a system (Cas13a) and catalytic hairpin assembly (CHA), Cas13a-CHA) and SERS-active silver nanorods (AgNRs) sensing chips was proposed for rapid and accurate detection of disease-related nucleic acids. Taking SARS-CoV-2 RNA assay as a model, the Cas13a-CHA based SERS sensing strategy can achieve ultra-high sensitivity low to 5.18 × 102 copies·mL−1 within 60 min, and excellent specificity, i.e., not only the ability to identify SARS-CoV-2 RNA from gene mutations, but also incompatibility with coronaviruses such as severe acute respiratory syndrome (SARS-CoV), Middle East respiratory syndrome (MERS-CoV), and other respiratory viruses. The proposed Cas13a-CHA based SERS assay for SARS-CoV-2 RNA has satisfactory sensitivity, specificity, uniformity, and repeatability, and can be easily expanded and universalized for screening different viruses, which is expected to promise as a crucial role for diagnosis of disease-related nucleic acids in various medical application scenarios.

Polymerized carbon dots with high electrochemiluminescence efficiency and long wavelength ECL emission for ultrasensitive detection of MicroRNA-222

Herein, a new electrochemiluminescence (ECL) biosensor was constructed with highly efficient polymerized carbon dots (PCDs) as ECL emitter and the improved localized catalytic hairpin assembly (L-CHA) as signal amplifier for ultrasensitive detection of microRNA-222 (miRNA-222). Impressively, compared to the traditional carbon dots with inefficient blue region ECL emission, PCDs with N, O co-dope and large conjugated π-system showed high electrical conductivity, narrow band gap and strong radiative transition, which could exhibit high ECL efficiency to improve the sensitivity of detection and long wavelength ECL emission to achieve deep tissue penetration for reducing biological damage. Furthermore, the trace target miRNA-222 could be efficiently converted into large amounts of output DNA labelled with the quencher dopamine (S-DA) through the L-CHA reaction to significantly enhance the target amplification efficiency for further improving the sensitivity of detection. Thus, the ECL biosensor could achieve the ultrasensitive detection of miRNA-222 from 100 aM to 100 pM with the detection limit of 76 aM. Therefore, this work proposed a novel CDs with high ECL efficiency and long wavelength ECL emission, which not only was used to build an ultrasensitive biosensor for biomolecules detection in clinical diagnosis, but also served as a potential emitter for ECL bioimaging.