The Compact Integration of Multiple Exonuclease III-Assisted Cyclic Amplification Units for High-Efficiency Ratiometric Electrochemiluminescence Detection of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) exhibits multiresistance to a plethora of antibiotics, therefore, accurate detection methods must be employed for timely identification to facilitate effective infection control measures. Herein, we construct a high-efficiency ratiometric electrochemiluminescent (ECL) biosensor that integrates multiple exonuclease (Exo) III-assisted cyclic amplification units for rapid detection of trace amounts of MRSA. The target bacteria selectively bind to the aptamer, triggering the release of two single-stranded DNAs. One released DNA strand initiates the opening of a hairpin probe, inducing exonuclease cleavage to generate a single strand that can form a T-shaped structure with the double strand connecting the oxidation-reduction (O-R) emitter of N-(4-aminobutyl)-N-ethylisoluminol gold (ABEI-Au). Consequently, ABEI-Au is released upon Exo III cleavage. The other strand unwinds the hairpin DNA structure on the surface of the reduction-oxidation (R-O) emitter ZIF-8@CdS, facilitating the subsequent release of a specific single strand through Exo III cleavage. This process effectively anchors the cathode-emitting material to the electrode. The Fe(III) metal-organogel (Fe-MOG) is selected as a substrate, in which the catalytic reduction of hydrogen peroxide by Fe(III) active centers accelerates the generation of reactive oxygen species and enhances signals from both ABEI-Au and ZIF-8@CdS. In this way, the two emitters cooperate to achieve bacterial detection at the single-cell level, and a good linear range is obtained in the range of 100-107 CFU/mL. Moreover, the sensor exhibited excellent performance in detecting MRSA across various authentic samples and accurately quantifying MRSA levels in serum samples, demonstrating its immense potential in addressing clinical bacterial detection challenges.

Cyclic Enzymatic Signal Amplification-Driven DNA Logic Nanodevices on Framework Nucleic Acid for Highly Sensitive Electrochemiluminescence Detection of Dual Myocardial miRNAs.

MicroRNAs (miRNAs) have emerged as promising biomarkers for acute myocardial infarction (AMI). There is an urgent imperative to develop analytical methodologies capable of intelligently discerning multiple circulating miRNAs. Here, we present a dual miRNA detection platform for AMI using DNA logic gates coupled with an electrochemiluminescence (ECL) response. The platform integrates DNA truncated square pyramids as capture probes on gold-deposited electrodes, enabling precise quantification of miRNA associated with AMI. The cyclic enzymatic signal amplification principle of strand displacement amplification enhances the miRNA detection sensitivity. AND and OR logic gates have been successfully constructed, enabling intelligent identification of miRNAs in AMI. Calibration curves show strong linear correlations between ECL intensity and target miRNA concentration (10 fM to 10 nM), with excellent stability in consecutive measurements. When applied to clinical serum samples, the biosensor exhibits consistent performance, underscoring its reliability for clinical diagnostics. This innovative approach not only demonstrates DNA nanotechnology's potential in biosensing but also offers a promising solution for improving AMI diagnosis and prognosis through precise miRNA biomarker detection.

Spectrally tunable humic acid-based carbon dots: a simple platform for metronidazole and ornidazole sensing in multiple real samples.

Humic acid-based carbon dots (HACDs) have excellent properties and are widely used in environmental detection, bioimaging, and optoelectronic materials. Herein, we investigated the structure-activity relationship between the morphology and optical properties of HACDs, and reported on a novel strategy for metronidazole (MNZ) and ornidazole (ONZ) sensing in multiple real samples. It was found that the average particle size decreased from 3.28 to 2.44 nm, optimal emission wavelength was blue-shifted from 500 to 440 nm, and the quantum yield (QY) improved from 5 to 23% with the temperature increasing from 110 to 400 °C. Under the oxidation of hydrogen peroxide (H2O2) and potassium permanganate (KMnO4), the UV-vis spectra of HACD aqueous solution showed time-dependent behavior, and the fluorescence emission of HACDs achieved spectrally tunable multi-color luminescence in the temporal dimension. The surface of HACDs contained a large number of hydroxyl (-OH) and carboxyl (-COOH) fluorophores, resulting in excellent pH sensing. Meanwhile, the synthesized HACDs revealed sensitive response to MNZ and ONZ with the limit of detection (LOD) of 60 nM and 50 nM in aqueous solutions, which had also been successfully applied in various actual samples such as lake water, honey, eggs, and milk with satisfactory results because of the inner filter effect (IFE). Our research is advantageous to enhance the potential applications of HACDs in advanced analytical systems.

Reversible Ratiometric Electrochemiluminescence Biosensor Based on DNAzyme Regulated Resonance Energy Transfer for Myocardial miRNA Detection.

Myocardial miRNAs in peripheral blood are closely related to the pathogenic process of myocardial infarction. Rapid identification and accurate quantification of myocardial miRNAs are of great significance to clinical interventions for treating cardiovascular lesions. Therefore, a ratiometric electrochemiluminescence (ECL) biosensor integrating DNAzyme with a resonance energy transfer (RET) system was designed to detect myocardial miRNA. The dual-signal system was composed of rA marked substrate strand functionalized CdTe quantum dots (QDs) as reductive-oxidative (R-O) emitters and Cy5-labeled strand-functionalized Ru(bpy)32+-filled silica nanoparticles (RuSi NPs) as oxidative-reductive (O-R) emitters. In the presence of target miRNA, DNAzyme was activated to cut substrate strands on the CdTe QDs and release triggers for opening hairpin probes. Then, the Cy5 molecule-labeled hairpin DNA on the RuSi NPs was opened to introduce Cy5 molecules and RuSi NPs into the system. The R-O ECL was quenched by ECL-RET between CdTe QDs and Cy5 molecules and the O-R ECL was introduced by the RuSi NPs. In this way, based on the simultaneous changing of the ECL signal, the dual-potential dynamic signal ratiometric ECL sensing platform was developed. By measuring the ratio of O-R ECL signal to R-O ECL signal, the concentration of miRNA-499 was accurately quantified in the range of 10 fM to 10 nM, and the detection limit was as low as 2.44 fM (S/N = 3). This DNAzyme guided dual-potential ratiometric ECL method provides a sensitive and reliable method for myocardial miRNA detection, and it has great potential in clinical diagnosis and treatment.

Three-Dimensional Surface-Enhanced Raman Scattering Platform with Hotspots Built by a Nano-mower for Rapid Detection of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the greatest threats to human health due to its strong drug resistance, wide distribution range, and high infection rate. Rapid identification of MRSA strains is very important for accurate diagnosis and early treatment of MRSA infections. Here, we introduced an Exo III-assisted nanomotor mower to build 3D hotspots for rapid detection of MRSA by surface-enhanced Raman scattering (SERS). As the bacteria bound to the aptamer, two trigger chains were released from the double-stranded structure, and the nano-mowers were activated by opening a hairpin probe on gold nanoparticles (AuNPs). With the continued cleavage of Exo III and cyclic release of the trigger chain, multiple hairpin DNAs on the AuNPs were cleaved to increase the motor power. The resulting nano-mower continued slicing protective DNA from larger AuNPs, exposing the AuNPs. Without the protection of DNA, Mg2+ in the buffer induced spontaneous aggregation of the AuNPs, and a large number of hotspots were formed for SERS measurements. Under optimal conditions, MRSA can be detected within 40 min, and the concentration of MRSA showed a good linear relationship with the SERS intensity at 1342 cm-1, with a limit of detection as low as 1 CFU/mL and a wide linear range (100 to 107 CFU/mL). This strategy creates a rapid bacterial detection method that performs well on actual samples utilizing portable Raman spectroscopy instruments, with potential applications in food detection, water detection, clinical treatment, and diagnosis.

Multistage nucleic acid amplification induced nano-aggregation for 3D hotspots-improved SERS detection of circulating miRNAs.

Circulating miRNAs in the blood can regulate disease development and thus indicate disease states via their various expression levels. For these reasons, circulating miRNAs constitute useful biomarkers, and an approach to the accurate detection of circulating miRNAs is attractive in the diagnosis and treatment of diseases. However, methods for clinical detecting of circulating miRNA that take both sensitivity and practicality into account are still needed. Therefore, we aimed herein to solve some inherent problems in the actual detection using a robust surface-enhanced Raman scattering (SERS) platform with integrated nucleic acid amplification and nanoparticle aggregation to construct 3D hotspots for improving performance of analyzing circulating miRNAs. After target recognition and initial signal amplification by DNAzyme, we observed that release triggered an open hairpin DNA on gold nanoparticles (AuNPs), which then promote AuNP aggregation, causing the accumulation of a large number of hotspots in three-dimention. The SERS biosensor achieved a better performance than the sandwich-type separation detection, with a low detection limit (0.37 fM) and a broad linear range (1 fM-10 nM) in liquids. This SERS platform can be used as a powerful tool for the detection of circulating miRNAs, and it can be used to improve the sensitivity and accuracy of various clinical-disease diagnoses.

A Multitargeted Electrochemiluminescent Biosensor Coupling DNAzyme with Cascading Amplification for Analyzing Myocardial miRNAs.

Several circulating miRNAs are associated with the pathogenic process of acute myocardial infarction (AMI). Thus, analyzing myocardial miRNAs in the circulatory system is important for the diagnosis and treatment of AMI, especially for early-stage diagnosis. Based on the characteristics of myocardial miRNAs, an ultrasensitive and multitargeted electrochemiluminescence (ECL) sensing platform was developed with a versatile probe that can couple DNAzyme with hybridization chain reaction amplification. The target miRNA and auxiliary chains form a circular unit that shears the versatile probe hairpin, and the products subsequently trigger cascading amplification; a long strand of dsDNA is then generated with many C-rich sequences that can undergo in situ reductions to generate ECL luminophore silver clusters. Using this strategy, three myocardial miRNAs are successfully detected with a detection limit as low as 29.6 aM (S/N = 3). Notably, our method can detect myocardial miRNA groups composed of multiple related circulating miRNAs with high selectivity over interfering miRNAs in blood. This is extremely important for solving the problem of diverse and low abundance of infarct-associated miRNAs. Our strategy pioneers a new idea of miRNA detection, and given its versatility and sensitivity, it is promising for the diagnosis of multigene-regulated cardiovascular diseases.

Band-pass filter-assisted ratiometric fluorescent nanoprobe composed of N-(2-aminoethyl-1,8-naphthalimide)-functionalized gold nanoclusters for the determination of alkaline phosphatase using digital image analysis.

A smartphone-based dual-wavelength digital imaging platform containing red (539-695 nm) and blue (389-511 nm) band-pass filters was developed for point-of-care (POC) testing of alkaline phosphatase (ALP) activity. The platform was based on dual-emitting fluorescent nanohybrids (AuNC@NAN), the ratiometric probe, which had a fluorescence "on-off-on-off" response. The probe comprised red-emitting gold nanoclusters (AuNCs) acting as the signal report units and blue-emitting N-(2-aminoethyl-1,8-naphthalimide) (NAN) acting as an internal reference. The different responses of the ratiometric probes resulted in a continuous color-multiplexing change from pink-red to dark-purple upon exposure to ALP. The dual-wavelength digital imaging platform was employed to acquire images of AuNC or NAN fluorescence signals without the influence of background light. Unlike the classical one-time digital imaging mode, the accurate red (R) and blue (B) channel values of the generated images can help to directly judge or eliminate the disturbance from unavoidable interfering factors. The R/B values were successfully employed for determining the ALP activity at a range 2.0 to 35.0 mU·mL-1 with the detection limit of 1.04 mU·mL-1. Such sensing imaging platform is also successful in determining ALP activity in human serum with 94.9-105% recoveries and relative standard deviation in the range 4.2-5.6%. A novel dual-wavelength smartphone-based digital imaging platform was proposed for simultaneous readout of the reporting and internal reference signals from dual-emitting ratiometric fluorescence probes, which allowed us to the accurate, reliable, and highly sensitive assay of ALP activity in complex samples.

Target-Induced Payload Amplification for Spherical Nucleic Acid Enzyme (SNAzyme)-Catalyzed Electrochemiluminescence Detection of Circulating microRNAs.

A high level of circulating myocardial microRNAs (miRNAs) is commonly considered as one indicator of acute myocardial infarction (AMI) for early stage diagnosis, and hence, it is of particular significance to develop ultrasensitive methodologies for detecting these miRNAs in circulating blood. Here we build an electrochemiluminescence (ECL) sensing platform for circulating miRNAs utilizing AuNPs@G-quadruplex (G4) spherical nucleic acid enzyme (SNAzyme) as the nanocatalyst, which shows good stability, strong nuclease resistance, and improved catalytic performance toward a luminol-H2O2 ECL system than the commonly used G4 DNAzyme. Target miRNA is employed to open the probe hairpin DNA to trigger the cascade amplification and then produce a long dsDNA chain with many sticky linkers that capture the SNAzyme nanocatalyst onto the electrode. In this way, two AMI-related miRNAs are detected accurately. The detection limit is 0.4 fM (S/N = 3), and the platform shows high selectivity in circulating blood. Given the good controllability, our ECL biosensors are promising for developing clinical diagnostic platforms for multiple indicators.

Spherical Nucleic Acid Enzyme (SNAzyme) Boosted Chemiluminescence miRNA Imaging Using a Smartphone.

As acute myocardial infarction (AMI) has now become a severe death threat to humans and may abruptly occur at home or outdoors where sophisticated equipment is not available, it is of great importance to develop facile methodologies for the point-of-care (POC) diagnosis of AMI. Toward this goal, here we build a sensing platform for chemiluminescence (CL) microRNA (miRNA) imaging with a smartphone as the portable detector, and for the first time we achieve visualization of AMI-related miRNAs in real patients' serum. We first construct a spherical nucleic acid enzyme (termed SNAzyme) derived from a dense layer of G-quadruplex (G4) DNAzyme formed on the gold nanoparticle core, which displays ∼100-fold and higher catalytic activity and improved resistance to nuclease degradation in a real blood sample as compared to those of the G4 DNAzyme itself. These unique features endow the SNAzyme-boosted CL platform with superior imaging performance for analyzing an AMI-related miRNA, miRNA-133a. This miRNA is employed to trigger the target-catalyzed hairpin assembly to produce a sticky dsDNA linker that captures the SNAzyme nanolabel onto the substrate. In this way, miRNA-133a is successfully detected, with a limit of detection of 0.3 pM (S/N = 3) and a high selectivity over other miRNA analogs in patients' blood. Given its unique features in physiological environments, our SNAzyme-boosted imaging platform holds great promise for use in the POC diagnosis of AMI.