Signal Amplification Strategy Research Articles

Highly efficient electrochemiluminescence based on Lu@Ov-ZMO as an ECL emitter for ultrasensitive detection of CA242

An effective electrochemiluminescence (ECL) emitter, luminol@oxygen vacancies-ZnMn2O4 (Lu@Ov-ZMO), was prepared to fabricate a novel luminol/H2O2-O2/Ov-ZMO ECL system for ultrasensitive detection of carbohydrate antigen 242 (CA242). The integrated design of co-reaction accelerator ZMO and luminol effectively shortened the electron transfer distance in the ECL system. Meanwhile, the abundant oxygen vacancies in ZMO further promoted the generation of more superoxide anion radicals (O2.-), which further improved the ECL luminescence efficiency. The resonance energy transfer (RET) strategy was used to construct the signal-off ECL immunesensor with PDA@SiO2 as the quencher. The fabricated immunosensor showed a good linear range of 0.01 mU/mL to 100 U/mL with a detection limit of 3.3 μU/mL (S/N = 3) under the optimum experimental conditions. This work put forward a new method of integrating luminophore and OVs enriched accelerator with wonderful signal-amplification strategy to formulate a functional immunosensor for early disease diagnostics.

Antibody-Bridged DNAzyme Walker for Sensitive Detection of Small Molecules.

Sensitive detection of small molecules with biological and environmental interests is important for many applications, such as food safety, disease diagnosis, and environmental monitoring. Herein, we propose a highly selective antibody-bridged DNAzyme walker to sensitively detect small molecules. The antibody-bridged DNAzyme walker consists of a track, small-molecule-labeled DNAzyme walking strand, and antibody against small molecules. The track is built by co-modifying fluorophore-labeled substrates and small-molecule-labeled DNA linkers onto a gold nanoparticle (AuNP). In the absence of the target molecule, the antibody binds small molecule labels at the DNAzyme walking strand and the DNA linker, driving the DNAzyme walking strand on the surface of the AuNP. The attached DNAzyme walking strand moves along the track and cleaves substrates to generate high fluorescence signals to achieve signal amplification. As target molecules exist, they competitively bind with antibody to displace the small-molecule-labeled linker and DNAzyme walking strand, rendering the DNAzyme walker inactive in substrate cleavage and causing weak fluorescence. By using this antibody-bridged DNAzyme walker, we achieved sensitive detection of two biologically important small molecules, digoxin and folic acid. This work provides a new paradigm by combining the signal amplification strategy of a DNA walker and immunorecognition for sensitive and selective detection of small molecules.

Light-harvesting iridium (III) complex-sensitized NiO photocathode for photoelectrochemical bioanalysis.

Anovel iridium (III) complex bearing boron dipyrromethene (Bodipy) as the light-harvesting antenna has been synthesized and is firstly employed as photosensitizer to assemble adye-sensitized NiO photocathode. The assembled photocathode exhibits significantly improved photoelectrochemical (PEC) performance. Integrating the prepared photocathode with hybridization chain reaction (HCR)--based signal amplification strategy, a cathodic PEC biosensor is proposed for the detection of microRNA-133a (miRNA-133a). In the presence of thetarget, HCR is triggered to form long duplex concatamers on the photocathode, which allows numerous manganese porphyrins (MnPP) to bind in the dsDNA groove. With the help of H2O2, MnPP with peroxidase-like activity catalyzes 4--chloro-1-naphthol (4-CN) to produce benzo--4--chlorohexadienone (4-CD) precipitate on the electrode, leading to a significant decrease of photocurrent signal. The decreased photocurrent correlates linearly with the target concentration from 0.1 fM to 1nM with a detection limit of 66.2 aM (S/N = 3). Theproposed PEC strategy exhibits delightful selectivity, reproducibility and stability.

Ratiometric fluorescence platform for the ultrasensitive detection of kanamycin based on split aptamer co-recognition triggers Mg2+-DNAzyme-driven DNA walker systems

In this work, a novel 3D-DNA walker signal amplification strategy was designed to construct a fluorescent aptasensor for the detection of kanamycin (KAN). The aptasensor utilizes split aptamers for the synergistic recognition of KAN. The presence of KAN induces the split aptamers recombination to form the Mg2+-DNAzyme structure, which is activated by Mg2+ to drive the 3D-DNA walker process for cascading signal amplification. Employing gold nanoflowers (AuNFs) as walking substrate material increases the local DNA concentration to enhance the walker efficiency. The prepared fluorescent aptasensor achieved efficient and sensitive detection of KAN with satisfactory results in the concentration range of 1 × 10−8 - 1 × 10−3 μg/kg and the detection limit of 5.63 fg/kg. Meanwhile, the designed fluorescent aptasensor exhibited favorable specificity, anti-interference, storage stability and reproducibility, and verified the feasibility of its application in milk samples. The present work provides an effective tool for the regulation of KAN contamination in animal-derived foods with promising prospects.

Artificial nanochannels for highly selective detection of miRNA based on the HCR signal amplification

Artificial solid-state nanochannels have nanoscale spatial confinement and unique ion transport properties that have been widely used in biosensing, nanofluidic devices, and nucleic acid sequencing. However, most of the nanochannel sensors for miRNA detection are grounded on the complementary pairing of bases, lacking signal amplification, which limits its application in many fields. Herein, we proposed a signal amplification strategy for miRNA-182 detection based on introducing hybridization chain reaction (HCR) in artificial nanochannels to improve the detection sensitivity. In this method, the capture DNA (cDNA) was anchored in the nanochannel, and when the target miRNA appeared, the hairpin probes H1, H2, and initiator were introduced to trigger the HCR reaction to form long nucleic acid double strands, which significantly enhanced the negative charge and affected ion transport in nanochannel. The nanochannel sensor performed excellent selectivity to the target miRNA-182, and it can realize the detection of miRNA-182 with an ultra-low concentration of 1.65 aM, which can be further applied to the detection of miRNA-182 in actual biological samples. Moreover, the nanochannel system was proved by using the AND logic gate that cDNA, initiator, H1, H2, and miRNA-182 are indispensable for successfully triggering HCR. This work provides a simple and efficient signal amplification method for miRNA detection, which considerably expands the application of nanochannel sensors and holds great prospects in biomedical and clinical detection.

A signal amplification strategy based on hexametaphosphate-stabilized BiVO4@CdS heterojunction with Mg2+-modulated surface charge for CEA detection

An ultrasensitive sensor with excellent photoelectrochemical (PEC) properties was developed based on the combination of electrostatic interactions between the hexametaphosphate (HMP)-coated BiVO4@CdS novel photoelectric material and the electron donor. Compared with the conventional BiVO4@CdS photoactive material, the HMP-modified BiVO4@CdS not only increases the stability, but also utilizes the electrostatic interactions between the negative charge carried by the phosphate group and the electron donor, which results to a significant increase in the photoelectric conversion ability after Mg2+ incubation. Specifically, carcinoembryonic antigen (CEA)-targeted immunorecognition was combined with MgCO3 antibody labelling inspired by the ability of HMP to complex with Mg2+. The released Mg2+ from the MgCO3 tag could neutralize the negative charge on the surface of BiVO4@CdS-HMP, and the electrostatic repulsion with the electron donor was suppressed, accelerating the electron transfer, and thus improving the electron-hole separation efficiency. A sensitive detection capability was demonstrated with CEA as the model target. Featuring a wide linear detection range with a detection limit as low as 60 fg/mL, this work provides a fresh perspective on the development of high-performance PEC biosensors.

Confinement-enhanced electrochemiluminescence by Ru(dcbpy)32+-functionalized γ-CD-MOF@COF-LZU1 porous hybrid material as micro-reactor for CYFRA 21-1 detection

The improvement of electrochemiluminescence (ECL) performance relies on the electron transfer efficiency between luminophore and coreactant. An ultrasensitive ECL micro-reactor with confinement-enhanced performance was prepared by using the covalent organic framework-LZU1-functionalized metal-organic framework (MOF@COF-LZU1) as a platform to assemble enormous N,N-dibutyl-2-hydroxyethylamine (DBAE) and tris(4,4′-dicarboxylic acid-2,2′-bipyridyl) ruthenium(II) [Ru(dcbpy)32+] into its pore channels. Compared to individual substances of γ-CD-MOF and COF-LZU1, the synergistic effects can conduce to the enhancement of the intensity, durability and sensitivity of the micro-reactor. Besides, COF-LZU1 can provide a mild environment to accommodate a certain amount of DBAE by concentrating them from the aqueous solution into its hydrophobic cavities and boost the oxidation efficiency of DBAE to generate more DBAE●+ and profited the survival of DBAE●, leading to an improved reaction efficiency with the Ru(dcbpy)32+ intermediate. Thanks to the confinement-enhanced strategy, engineered as high-functioning luminescent materials, Ru@γ-CD-MOF@COF-LZU1 micro-reactors decorated with Au NPs can facilitate electron transfer and capture primary antibodies (Ab1). Moreover, Au–Pd–Pt noble metal aerogels (NMAs) functionalized MoS2 NFs (Au–Pd–Pt NMAs@MoS2 NFs) were chosen as base material due to its large specific surface areas, high porosity, and excellent electrical conductivity. Based on above merits, the sensor demonstrated a sensitive response to CYFRA 21-1 detection in a linear concentration gradient from 10 fg/mL to 50 ng/mL with a detection limit of 0.0055 pg/mL (S/N = 3). The COF-LZU1 decorated ECL micro-reactors were constructed based on the signal amplification strategies to realize accurate CYFRA 21-1 detection.

Design and analysis of self-priming extension DNA hairpin probe for miRNA detection based on a unified dynamic programming framework

MicroRNAs (miRNAs) are potential biomarkers for cancer diagnosis and prognosis, methods for detecting miRNAs with high sensitivity, selectivity, and stability are urgently needed. Various nucleic acid probes that have traditionally been for this purpose suffer several drawbacks, including inefficient signal-to-noise ratios and intensities, high cost, and time-consuming method establishment. Computing tools used for investigating the thermodynamics of DNA hybridization reactions can accurately predict the secondary structure of DNA and the interactions between DNA molecules. Herein, NUPACK was used to design a series of nucleic acid probes and develop a phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) signal amplification strategy, which enabled the ultrasensitive detection of miR-200a in serum samples. The free and binding energies of the DNA detection probes calculated using NUPACK, as well as the biological experimental results, were considered synthetically to select the best sequence and experimental conditions. A unified dynamic programming framework, NUPACK analysis and the experimental data, were complementary and improved the designed model in all respects. Our study demonstrates the feasibility of using computer technology such as NUPACK to simplify the experimental process and provide intuitive results.

A novel split-type photoelectrochemical biosensor based on liposome-induced in situ formation of Bi2S3@BiOI/Ag2S heterojunction for the sensitive detection of CEA

A novel split-type photoelectrochemical (PEC) biosensor was constructed for the sensitive detection of carcinoembryonic antigen (CEA) based on the Ag+-liposome (ALL) signal amplification strategy and Bi2S3@BiOI heterojunction as the substrate material. The split-type sensing mode effectively avoids the complex fixation of biomolecules on the electrode surface and preserves the activity of biomolecules. The sandwich complex was formed between CEA and aptamer-anchored streptavidin-functionalized magnetic beads (SMB-Apt) and ALL-labeled aptamer (ALL-Apt). After the methanol was added, a large amount of Ag+ was released from the liposome. The released Ag+ was transferred to the Bi2S3@BiOI modified electrode surface for incubation and then soaked in Na2S solution to form a narrow band gap photosensitive material Ag2S on the electrode surface. Bi2S3@BiOI/Ag2S ternary heterojunction in situ was formed due to the band gap matching relationship between Ag2S and the Bi2S3@BiOI heterojunction, which effectively expanded the light absorption range and enhanced the photocurrent signal. Under the optimal experimental conditions, the constructed PEC sensing platform exhibited good analytical performance for CEA with a linear range of 0.005 ∼ 50 ng mL−1 and a detection limit of 1.21 pg mL−1. Additionally, the PEC sensor had good selectivity, reproducibility, and stability, which provided a new research idea for the detection of clinical tumor biomarkers.

Label-Free, Sensitive, and Versatile Colorimetric Method for Molecule Detection via the G-Quadruplex-Based Signal Quenching Strategy.

Signal amplification strategies have emerged as a prominent tool in the field of improving the detection sensitivity of small extracellular vesicles (sEVs). It is important to highlight that the utilization of signal quenching strategies is not commonly implemented. A detection technique for sEVs was established based on the unwinding of G-quadruplex using Klenow fragment polymerase (KF), which served as an inspiration for this study. This system is characterized by its simplicity and lack of labeling, making it an efficient approach for signal quenching. In the presence of sEVs, the CD63 aptamer in the capture@sMBs complex binds with the CD63 protein on the surface of sEVs to release trigger sequences, which were employed as a primer to mediate the DNA polymerase/endonuclease-assisted signal recycling. The signal recycling process produces numerous single-stranded DNA sequences that can bind to the toehold section of the G-quadruplex. This leads to the rupture of the G-quadruplex structure and the subsequent deactivation of a DNAzyme generated by the G-quadruplex structure and hemin, thereby inhibiting its biological catalytic function. Consequently, the G-quadruplex structure would undergo a transformation to a duplex structure, leading to the emergence of a discernible differential signal that can be noticed in a majority of instances, even without the aid of magnification devices. The decrease in the prominent signal allows for the efficient analysis of target sEVs, which exhibit a notably low detection limit. In addition to the detection of sEVs, the approach has also been utilized for the investigation of miRNA-21. The approach demonstrates a high level of selectivity and robustness in its capacity to differentiate between target miRNA and base-mismatched miRNA as well as other miRNA families. This statement suggests that the assay holds significant promise for use in biochemical research and clinical diagnosis.

“Two in one”: A novel DNA cascade amplification strategy for trace detection of dual targets

According to the characteristics of DNA programming, the cascaded nucleic acid amplification technology with larger output can overcome the problem of insufficient sensitivity of single nucleic acid amplification technology, and it combines the advantages of two or even multiple nucleic acid amplification technologies at the same time. In this work, a novel cascade signal amplification strategy with strand displacement amplification (SDA) and cascade hybridization chain reaction (HCR) was proposed for trace detection of hAAG and VEGF165. HAAG-induced SDA produced a large amount of S2 to open H2 on Polystyrene (PS) nanospheres, thereby triggering cascade HCR to form DNA dendritic nanostructures with rich fluorescence (FL) signal probes (565 nm). It could realize the amplification of FL signals for the detection of hAAG. Moreover, many doxorubicin (Dox) were loaded into the GC bases of DNA dendritic nanostructures, and its FL signal was effectively shielded. VEGF165 specifically bound to its aptamer to form G-quadruplex structures, which released Dox to produce a high FL signal (590 nm) for detection of VEGF165. This work developed a unique multifunctional DNA dendritic nanostructure fluorescence probe, and cleverly designed a new “On-off” switch strategy for sensitive trace detection of cancer markers.

Binding-induced DNA assembly makes Exo III-aided signal amplification universal for highly sensitive protein detection

Exonuclease III has been involved in many signal amplification strategies for bioanalysis. Although Exo III-based signal amplification methods are sensitive and simple compared to other nuclease-based strategies, existing methods do not work for all proteins. In this work, we combine Exo III-aided signal amplification with binding-induced DNA assembly for highly sensitive protein detection. Two separate single-stranded DNA will hybridize after binding to the same target, and trigger the Exo III-aided target recycling amplification. This strategy can realize homogeneous detection of all proteins, and the detection conditions and performance were discussed. Human epididymis protein-4 (HE4) was token as example, a linear detection range of 0.01 ∼ 50 ng/mL, with the LOD of 3 pg/mL for rapid detection is presented. Notably, a linear relationship in range of 0.05 ∼ 1 pg/mL can be obtained with long time incubation. In conclusion, we believe that this novel approach will have significant implications in life science research, disease diagnosis, treatment, and prognosis.

Exploring Simple Particle-Based Signal Amplification Strategies in a Heterogeneous Sandwich Immunoassay with Optical Detection.

Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current "gold standard" enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidin-functionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C-reactive protein (CRP), the analyte sensitivity achievable with optimized PSP systems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.

Sandwich-type electrochemical aptasensor based on hemin-graphite oxide as a signal label and rGO/MWCNTs/chitosan/carbon quantum dot modified electrode for sensitive detection of Acinetobacter baumannii bacteria

Acinetobacter baumannii (A. baumannii) is a pathogenic bacterium that causes severe infections and its rapid and reliable diagnosis is essential for effective control and treatment. In this study, we present an electrochemical aptasensor based on a signal amplification strategy for the detection of A. baumannii, the high specificity and affinity of the aptamer for the target make it favorable for signal amplification. This allows for a highly sensitive and selective detection of the target. The aptasensor is based on a carbon screen-printed electrode (CSPE) that has been modified with a nanocomposite consisting of multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (rGO), chitosan (CS), and a synthesized carbon quantum dot (CQD) from CS. Additionally, the self-assembled aptamers were immobilized on hemin-graphite oxide (H-GO) as a signal probe.The composition of the nanocomposite (rGO-MWCNT/CS/CQD) provides high conductivity and stability, facilitating the efficient capture of A. baumannii onto the surface of the aptasensor. Also, aptamer immobilized on Hemin-graphite oxide (H-GO/Aptamer) was utilized as an electrochemical signal reporter probe by H reduction. This approach improved the detection sensitivity and the aptamer surface density for detecting A. baumannii. Furthermore, under optimized experimental conditions, the aptasensor was demonstrated to be capable of detecting A. baumannii with a linear range of (10 – 1 × 107 Colony-forming unit (CFU)/mL) and a limit of detection (LOD) of 1 CFU/mL (σ = 3). One of the key features of this aptasensor is its ability to distinguish between live and dead bacteria cells, which is very important and critical for clinical applications. In addition, we have successfully detected A. baumannii bacteria in healthy human serum and skim milk powder samples provided using the prepared electrochemical aptasensor.The functional groups present in the synthetic CQD, rGO-MWCNT, and chitosan facilitate biomolecule immobilization and enhance stability and activity. The fast electron-transfer kinetics and high conductivity of these materials contribute to improved sensitivity and selectivity. Furthermore, The H-GO/Aptamer composite's large surface area increases the number of immobilized secondary aptamers and enables a more stable structure. This large surface area also facilitates more H loading, leading to signal amplification.

Self-enhanced nanohydrogel electrochemiluminescence biosensor based on CRISPR/Cas12a and gold platinum nanoparticles modification for high-sensitivity detection of Burkholderia pseudomallei

The simple, rapid, and accurate detection of highly lethal melioidosis is crucial for early clinical diagnosis and improving cure rates. Currently, due to time consumption, low sensitivity and detection rate the existing clinical detection methods cannot satisfy the needs of clinical diagnosis. Herein, a novel self-enhanced porous hydrogel material (Au@PEI-ABEI@Pt) for ultrasensitive ECL strategy of detection B. pseudomallei was report. The novel porous hydrogel material composed of PEI-ABEI porous hydrogel, gold nanoparticles (AuNP) and platinum nanoparticles (PtNP) have large specific surface area and porous structure, which not only fix more ABEI to realize self-enhanced ECL signal amplification, but also facilitate ion diffusion and efficient utilization of catalytic materials, realizing rapid electron transfer and zero-distance catalysis, significantly improving the initial signal of ECL sensor. Moreover, the most attractive aspect is that Au@PEI-ABEI@Pt hydrogels with good biocompatibility can achieve widespread application of CRISPR/Cas12a in solid-phase carriers without affecting the sensitivity, specificity and shearing activity of CRISPR/Cas12a. After coupling with the ECL system and CRISPR/Cas12a signal amplification strategy, the Au@PEI-ABEI@Pt can achieve an ultrasensitive ECL assay of B. pseudomallei with the LOD of 5 CFU mL−1 in complex samples, with high specificity and stability to effectively classify B. pseudomallei and other Gram-negative bacteria. This study shows that the developed porous hydrogel materials not only serve as an excellent ECL signal reporter to significantly improve the detection sensitivity of ECL biosensors, but also provide a new approach for the wide application of CRISPR/Cas systems in solid-phase carriers.

Spatial High-resolution Analysis of Gene Expression Levels in Tendons.

In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich tissues, and specifically, tendons were left behind due to their low cell number, low RNA amount per cell, and high matrix content, which made them complicated to analyze. One of the recent and most important single-cell tools is the spatial analysis of gene expression levels in tendons. These RNA spatial tools have specifically high importance in tendons to locate specific cells of new and unknown populations, validate single-cell RNA-seq results, and add histological context to the single-cell RNA-seq data. These new methods will enable the analysis of RNA in cells with exceptional sensitivity and the detection of single-molecule RNA targets at the single-cell level, which will help to molecularly characterize tendons and promote tendon research. In this method paper, we will focus on the available methods to analyze spatial gene expression levels on histological sections by using novel in situ hybridization assays to detect target RNA within intact cells at single-cell levels. First, we will focus on how to prepare the tendon tissue for the different available assays and how to amplify target-specific signals without background noise but with high sensitivity and high specificity. Then, the paper will describe specific permeabilization methods, the different probe designs, and the signal amplification strategies currently available. These unique methods of analyzing transcription levels of different genes in single-cell resolution will enable the identification and characterization of the tendon tissue cells in young and aged populations of various animal models and human tendon tissues. This method will also help analyze gene expression levels in other matrix-rich tissues such as bones, cartilage, and ligaments.

Dual-source signal amplification electrochemiluminescence sensor combined with molecularly imprinted polymers for the imidacloprid detection

A novel lanthanide metal-organic-gel (MOG)-derived material/nitrogen-doped graphdiyne (Tb-Ru-MOG/CeO2/N-GDY) composite with a dual-source signal amplification strategy was prepared and used to construct a molecularly imprinted sensor based on bifunctional monomers for the detection of imidacloprid (IMI) using electrochemiluminescence (ECL). In a green reaction environment, terbium (III) (Tb3+) can undergo multiple coordination reactions with 4′-(4-carboxyphenyl)-2,2′:6′,2″-terpyridine (Hcptpy) and tris(4,4′-dicarboxylicacid-2,2′-bipyridyl) ruthenium (II) dichloride (Ru(dcbpy)32+), and combine with ceria nanoparticles (CeO2 NPs) to form Tb-Ru-MOG/CeO2. Within the Tb-Ru-MOG/CeO2 framework, energy transfer from the double ligands can sensitize the central Tb3+, triggering a distinct antenna effect and energy-transfer, and its polyporous configuration offered a nanoconfined space for Ce3+/Ce4+ to effectively catalyze coreactant radicals (S2O82−), leading to in-situ endogenous activation ECL reactions. The conductive N-GDY accelerated electron movement and increased the loading on the electrode surface, enhancing the exogenous excitation of the ECL signals. Leveraging the synergistic effect of the bifunctional monomer, the synthesized molecularly imprinted polymers (MIPs) ECL sensor demonstrated a wide detection range from 10 nM to 10,000 nM for IMI, with a limit of detection (LOD) of 1.37 nM, showcasing an innovative concept for the dual-source strategy of signal amplification in integrated ECL composites to analyze food and environmental hazards.

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.

Multiple signal amplification strategy induced by biomarkers of lung cancer: A self-powered biosensing platform adapted for smartphones

Lung cancer is a major malignant cancer with low survival rates, and early diagnosis is crucial for effective treatment. Herein, a biosensing platform that is self-powered derived from a capacitor-coupled EBFC has been developed for ultra-sensitive real-time identification of microRNA-21 (miRNA-21) with the assistance of a mobile phone. The flexible substrate of the platform is prepared on a carbon paper modified with graphdiyne and gold nanoparticles. The biosensor employs DNAzyme-mediated dual strand displacement amplification, which enhances the signal output intensity of the EBFC and improves selectivity. The coupling of the capacitor with the EBFC significantly amplifies the sensing signal, causing a 10.6-fold surge in current respond and further improving the sensitivity of the sensing platform. The established detection approach demonstrates a linear relationship varied from 0.0001 to 10,000 pM, with a sensitivity down to 32.3 aM as the minimum detectable limit, which has been effectively utilized for detecting miRNA-21 in practical samples. This sensing system provides strong support for the construction of portable detection devices, and the strategy of the platform construction provides an effective method for ultra-sensitive and accurate detection of miRNA, holding great potential in clinical diagnosis, prognosis evaluation, and drug screening for cancer.

Ultrasensitive ECL immunosensor for trenbolone detection based on double-assisted signal amplification strategy of Nb2O5-decorated TiO2 nanorods and Co3O4-Co3(PO4)2 nanosheets

Trenbolone (TB) is used for treating malnutrition in livestock and increasing their muscle mass. However, its excessive intake can lead to metabolic and reproductive disorders in animals. In addition, it causes adverse effects in humans even at very low concentrations. Herein, an innovative electrochemiluminescence (ECL) immunosensor based on a double-assisted signal amplification strategy has been proposed for the sensitive detection of TB. Nb2O5-decorated TiO2 nanorods, with a large surface area, good biocompatibility, and excellent catalytic properties, were used as the substrates for the effective loading of TB antibodies. In addition, Co3O4-Co3(PO4)2 nanosheets, used as labels, improved the ECL signals and were used to incorporate TB antigens for designing competitive ECL immunosensors. Various experimental factors, such as the pH of the phosphate buffer solution, concentration of the co-reactant, and time of incubation, were examined. The proposed ECL immunosensor exhibited high sensitivity, good repeatability, and high stability for TB concentration and was successfully implemented for the analysis of real water samples. Under optimal conditions, the proposed immunosensor responded linearly to relevant concentrations of TB from 1.0 × 10−3 to 1.0 × 103 ng/mL, with a detection limit of 0.39 × 10−3 ng/mL (S/N = 3). Notably, this study fabricated a simple and ultrasensitive TB detection system with novelty of practical application in environmental monitoring.