Signal Amplification Research Articles

Electron-Accelerator-Induced Fast Electron Transfer for Enhancing Electrochemiluminescence of Gold Nanoclusters and Its Bioanalysis Application: A Novel Avenue for Developing High-Efficient Emitters.

Herein, the gold nanoclusters/CaFe2O4 nanospheres (Au NCs/CaFe2O4) heterostructure as a novel electrochemiluminescence (ECL) emitter was developed. Excitingly, Au NCs/CaFe2O4 displayed highly efficient and greatly stable ECL based on the newly defined electron-accelerator p-type semiconductor CaFe2O4 NS-induced fast electron transfer; it solved one key obstacle of metal NC-based ECL emitters: sluggish through-covalent bond electron transport kinetics-caused inferior ECL performance. Specifically, on account of the energy level matching between emitter Au NCs and electron-accelerator CaFe2O4 NSs, the valence band (VB) of the electron-accelerator could provide abundant holes for rapidly transporting the electrogenerated electron from the highest occupied molecular orbital (HOMO) of Au NCs to the electrode, generating massive excited species of Au NCs for strong ECL emission. Notably, Au NCs/CaFe2O4 emerged 5.4-fold higher ECL efficiency with 3.5-fold higher electrochemical oxidation current in comparison with pure Au NCs, exhibiting great prospects in extensive lighting installations, ultrasensitive biosensing, and high-resolution ECL imagery. As applications, an ECL bioassay platform was constructed with Au NCs/CaFe2O4 as an emitter and U-like structure-fueled catalytic hairpin assembly (U-CHA) as a signal amplifier for fast and trace analysis of aflatoxin B1 (AFB1) with the detection limit (LOD) down to 2.45 fg/mL, which was 3 orders of magnitude higher than that of the previous ECL biosensors with much better stability. This study developed an entirely new avenue for enlarging the ECL performance of metal NCs, and it is a very attractive orientation for directing the reasonable design of prominent metal NC-based ECL emitters and broadening the practical application of metal NCs.

Dual-Function DNA Nanowires with Self-Feedback Amplification and Efficient Signal Transduction for Intracellular Imaging of MicroRNA-155.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

High-Performance Suspension Bead Sensor Based on Optical Tweezers and Immuno-Rolling Circle Amplification.

In recent years, optical tweezers have become an effective bioassay tool due to their unique advantages, especially in combination with suspension beads, which can be applied to develop a high-performance analysis platform capable of high-quality imaging and stable signal output. However, the optical tweezer-assisted bead analysis is still at the early stage, and further development of different favorable methods is in need. Herein, we have first developed the optical tweezer-assisted immuno-rolling circle amplification (immuno-RCA) on beads for protein detection. Prostate-specific antigen was selected as the model analyte, and the immunosandwich structure on beads was built by the high affinity of "antibody-antigen". The "protein-nucleic acid" signals were effectively converted through the covalent coupling procedure of antibodies and oligonucleotides, further initiating the RCA reaction to achieve signal amplification. The individual beads with the strong irregular Brownian motion in a fluid environment were eventually trapped by the optical tweezers to acquire the accurate and high-quality signal. Compared with the conventional immunoassay on beads, the sensitivity of the developed strategy was increased by 587 times with a limit of detection of 4.29 pg/mL (0.13 pM), as well as excellent specificity, stability, and reproducibility. This study developed the new optical tweezer-assisted beads imaging strategy for protein targets, which has great potential for being applied to clinical serology research and expands the application of optical tweezers in the bioassays.

Polypyrrole deposited on the core-shell structured nitrogen-doped porous carbon@Ag-MOF for signal amplification detection of chloride ions.

An electrochemical platform for signal amplification probing chloride ions (Cl-) is constructed by the composite integrating core-shell structured nitrogen-doped porous carbon@Ag-based metal-organic frameworks (NC@Ag-MOF) with polypyrrole (PPy). It isbased on the signal of solid-state AgCl derived from Ag-MOF, sinceboth NC and PPy have good electrical conductivityand promote the electron transport capacity of solid-state AgCl. NC@Ag-MOF was firstly synthesized with NC as the scaffold and then, PPy was anchored on NC@Ag-MOF by chemical polymerization. The composite NC@Ag-MOF-PPy was utilized to modify theelectrode, which exhibited a higher peak current and lower peak potential during Ag oxidation compared with those of Ag-MOF and NC@Ag-MOF-modified electrodes. More importantly, in the coexistence of chloride (Cl-) ions in solution, theNC@Ag-MOF-PPy-modified electrode displayed a fairly stable and sharp peak of solid-state AgCl with the peak potentials gradually approaching zero, which might effectively overcome the background interference caused by electroactive substances. Theoxidation peak currents of solid-state AgCl increased linearly with the concentrationof Cl- ions in a broad range of 0.15µM-40mM and 40-250mM, with detection limits of 0.10µM and 40mM, respectively. Thepractical applicability for Cl- ions determinationwas demonstrated usinghuman serum and urinesamples. Theresults suggest that NC@Ag-MOF-PPy composite could be a promising candidate for the construction of the electrochemical sensor.

The potential of ALFA-tag and tyramide-based fluorescence signal amplification to expand the CRISPR-based DNA imaging toolkit.

Understanding the spatial organization of genomes within chromatin is crucial for deciphering gene regulation. A recently developed CRISPR-dCas9-based genome labeling tool, known as CRISPR-FISH, allows efficient labelling of repetitive sequences. Unlike standard fluorescence in situ hybridization (FISH), CRISPR-FISH eliminates the need for global DNA denaturation, allowing for superior preservation of chromatin structure. Here, we report on the further development of the CRISPR-FISH method, which has been enhanced for increased efficiency through the engineering of a recombinant dCas9 protein containing an ALFA-tag. Using an ALFA-tagged dCas9 protein assembled with an A. thaliana centromere-specific gRNA, we demonstrate target-specific labelling with a fluorescence-labeled NbALFA nanobody. The dCas9 protein possessing multiple copies of the ALFA-tag, in combination with a minibody and fluorescence-labelled anti-rabbit secondary antibody, resulted in enhanced target-specific signals. The dCas9-ALFA-tag system was also instrumental in live cell imaging of telomeres in N. benthamiana. This method will further expand the CRISPR imaging toolkit, facilitating a better understanding of genome organization. Furthermore, we report the successful integration of the highly sensitive Tyramide Signal Amplification (TSA) method with CRISPR-FISH, demonstrating effective labeling of A. thaliana centromeres.

High‐Performance Cooperative DNA Nanodevice Enables Sensitive Circular RNA Imaging and Precise Tumor Growth Suppression

The utility of circular RNAs (circRNAs) as emerging biomarkers and regulatory factors in medical diagnostics and therapeutics is hampered by the challenges associated with their sensitive detection and precise modulation. Herein, a high‐performance cooperative DNA nanodevice (HCDN) based on DNA tetrahedron‐confined catalytic DNA assembly reaction (DT‐CDA) that enables both imaging and regulation of circRNAs is developed. Activation of the DT‐CDA is contingent upon the presence of the target circRNA, which, together with a replicative fuel probe, catalyzes the sequential opening of additional DT‐CDAs. This cooperative exponential signal amplification with negligible background interference allows HCDN to effectively detect minute quantities of circRNAs. Employing circSATB2 as a model, the HCDN demonstrates substantial downregulation of Cyclin D1 (CCND1) mRNA and protein levels in cellular and in vivo models, thereby inhibiting tumor growth. The innovative design of HCDN sets the stage for a powerful methodology conducive to enhanced clinical diagnostics and biomolecule manipulation, thereby advancing the capabilities and applications of DNA nanotechnology.

A Systems Biology Analysis of Chronic Lymphocytic Leukemia

Whole-genome sequencing has revealed that TP53, NOTCH1, ATM, SF3B1, BIRC3, ABL, NXF1, BCR, and ZAP70 are often mutated in CLL, but not consistently across all CLL patients. This paper employs a statistical thermodynamics approach in combination with the systems biology of the CLL protein–protein interaction networks to identify the most significant participant proteins in the cancerous transformation. Betti number (a topology of complexity) estimates highlight a protein hierarchy, primarily in the Wnt pathway known for aberrant CLL activation. These individually identified proteins suggest a network-targeted strategy over single-target drug development. The findings advocate for a multi-target inhibition approach, limited to several key proteins to minimize side effects, thereby providing a foundation for designing therapies. This study emphasizes a shift towards a comprehensive, multi-scale analysis to enhance personalized treatment strategies for CLL, which could be experimentally validated using siRNA or small-molecule inhibitors. The result is not just the identification of these proteins but their rank-order, offering a potent signal amplification in the context of the 20,000 proteins produced by the human body, thus providing a strategic basis for therapeutic intervention in CLL, underscoring the necessity for a more holistic, cellular, chromosomal, and genome-wide study to develop tailored treatments for CLL patients.

Signal Amplification By Reversible Exchange and its Translation to Hyperpolarized Magnetic Resonance Imaging in Biomedicine

Hyperpolarized magnetic resonance imaging (HP‐MRI) has emerged as a powerful tool in molecular imaging, providing in vivo, real‐time insights into metabolic pathways without ionizing radiation. Signal Amplification by Reversible Exchange (SABRE) represents a promising hyperpolarization technique, leveraging parahydrogen to enhance MRI signals. In this concept, we delineate the evolution of SABRE and landmark papers that have enabled us recently to produce biocompatible and low‐cost hyperpolarized pyruvate within minutes for in vivo metabolic imaging, showcasing SABRE’s potential for preclinical and near‐future clinical settings. Looking ahead, with ongoing efforts focused on optimizing polarizer technology and expanding applications beyond pyruvate, we envision SABRE as a key player in the research and application of HP‐MRI due to its simplicity and throughput.

Signal-On Detection of Caspase-3 with Methylene Blue-Loaded Metal-Organic Frameworks as Signal Reporters

In this work, we report on an electrochemical method for the signal-on detection of caspase-3 and the evaluation of apoptosis based on the biotinylation reaction and the signal amplification of methylene blue (MB)-loaded metal–organic frameworks (MOFs). Zr-based UiO-66-NH2 MOFs were used as the nanocarriers to load electroactive MB molecules. Recombinant hexahistidine (His6)-tagged streptavidin (rSA) was attached to the MOFs through the coordination interaction between the His6 tag in rSA and the metal ions on the surface of the MOFs. The acetylated peptide substrate Ac-GDEVDGGGPPPPC was immobilized on the gold electrode. In the presence of caspase-3, the peptide was specifically cleaved, leading to the release of the Ac-GDEVD sequence. A N-terminal amine group was generated and then biotinylated in the presence of biotin-NHS. Based on the strong interaction between rSA and biotin, rSA@MOF@MB was captured by the biotinylated peptide-modified electrode, producing a significantly amplified electrochemical signal. Caspase-3 was sensitively determined with a linear range from 0.1 to 25 pg/mL and a limit of detection down to 0.04 pg/mL. Further, the active caspase-3 in apoptosis inducer-treated HeLa cells was further quantified by this method. The proposed signal-on biosensor is compatible with the complex biological samples and shows great potential for apoptosis-related diagnosis and the screening of caspase-targeting drugs.

Recent Advances in Aptamer‐Based Sensors for In Vitro Detection of Small Molecules

AbstractSensitive and accurate detection of small molecules from complex matrix has aroused increasing interest in many fields, yet remains an open challenge. Recent years have witnessed a considerable advance of aptasensors for diagnostic assay development towards diverse small molecules because aptamer is one of the most powerful classes of molecular receptors with advanced affinity and specificity. Herein, we reviewed the small‐molecule aptasensors in the past five years, focusing on the principles to specific applications in clinical diagnosis, food safety, and environmental monitoring. The first introductory section on the development of aptasensors in historical view and its analytical features contextualizes essential health‐related small molecules. The second part highlights the basic components of aptasensor and the detection principles of different sensors based on signal output modes. The subsequent part systematically discusses various small‐molecule sensing platforms by interfacing aptamers with diverse signal amplification strategies. Finally, challenges and perspectives for improving the aptasensor performance are also discussed. By describing biochemical and analytical procedures, this review highlights the optimal use of aptamers in the detection, quantification, and imaging of important health‐related small molecules and presents new insights, technical advances, and engineering strategies for practical applications.

Engineering an upconversion fluorescence sensing platform with "off-on" pattern through specific DNAzyme-mediated signal amplification for supersensitive detection of uranyl ion.

Anupconversion fluorescence sensing platform was developedwith upconversion nanoparticles (UCNPs) as energy donors and gold nanoparticles (AuNPs) as energy acceptors, based on the FRET principle. They were used for quantitativedetection ofuranyl ions (UO22+) by amplifying the signal of the hybrid chain reaction (HCR). When UO22+ are introduced, the FRET between AuNPs and UCNPs can be modulated through a HCR in the presence of high concentrations of sodium chloride. This platform providesexceptional sensitivity, with a detection limit as low as 68pM for UO22+ recognition. We have successfully validated the reliability of this method by analyzing authentic water samples, achieving satisfactory recoveries (89.00%-112.50%) that are comparable to those of ICP-MS. These results indicate that the developed sensing platform has the capability to identify trace UO22+ in complex environmentalsamples.

Sesame Detection in Food Using DNA-Functionalized Gold Nanoparticles: A Sensitive, Rapid, and Cost-Effective Colorimetric Approach

Food safety control is a key issue in the food and agriculture industries. For such purposes, developing miniaturized analytical methods is critical for enabling the rapid and sensitive detection of food supplements, allergens, and pollutants. Here, a novel bioanalytical methodology based on DNA-functionalized gold nanoparticles (AuNPs) and colorimetric detection was developed to detect the presence of sesame (a major allergen) through sesame seed DNA as a target, in food samples. The presence of sesame DNA induces controlled nanoparticle aggregation/desegregation, resulting in a color change (from blue to red) proportional to sesame DNA concentration. The incorporation of multicomponent nucleic acid enzymes (MNAzymes) in this strategy has been carried out to perform an isothermal signal amplification strategy to improve the sensitivity of detection. Also, open-source software for color analysis was used to ensure an unbiased visual color-change detection, enhancing detection accuracy and sensitivity and opening the possibility of performing a simple and decentralized analyte detection. The method successfully detected the presence of sesame DNA in sesame seed, sesame oil, olive oil, and sunflower oil. In brief, the developed approach constitutes a simple and affordable alternative to perform a highly sensitive detection of DNA in food without complex methodologies or the requirement of expensive instrumentation.

A dual-signal mode electrochemical aptasensor based on tetrahedral DNA nanostructures for sensitive detection of citrinin in food using PtPdCo mesoporous nanozymes

Citrinin (CIT) is a mycotoxin with nephrotoxicity and hepatotoxicity, presenting a significant threat to human health that is often overlooked. Therefore, a dual-signal mode (DPV and SWV) aptasensor for citrinin (CIT) detection was constructed based on tetrahedral DNA nanostructures (TDN) in this study. Furthermore, PtPdCo mesoporous nanozymes exhibit catalase-like catalytic functions, generating significant electrochemical signals through a Fenton-like reaction. Meanwhile their excellent Methylene Blue (MB) loading capability ensures independent dual signal outputs. The RecJf exonuclease-assisted (RecJf Exo-assisted) process can expand the linear detection range, enabling further amplification of the signal. Under optimized conditions, the constructed aptaensor exhibited excellent detection performance with limits of detection (LODs) of 7.67 × 10−3 ng·mL−1 (DPV mode) and 1.57 × 10−3 ng·mL−1 (SWV mode). Due to its multiple signal amplification and highly accurate dual-signal mode detection capability, this aptasensor shows promising potential for the in situ detection.

Three-way junction-mediated three-letter coded SDA cascade CRISPR/Cas12a system for circRNA detection

Circular RNAs (circRNAs), a special type of non-coding RNAs with covalently closed circular structure, have emerged as promising liquid biopsy biomarker. However, the precise detection of circRNAs is severely hampered by interference from homologous linear RNAs, posing a significant obstacle for their applications in molecular diagnostics. Herein, a strategy named three-way junction (TWJ)-mediated three-letter coded strand displacement amplification (SDA) cascade CRISPR/Cas12a system (TTSC) has been devised to achieve precise detection of circRNAs. Three-letter coded SDA (TC-SDA) has been originally designed in this study for eliminating undesired interference from linear RNA and performing dual functionality in both discrimination and amplification. With this design, the proposed strategy elegantly distinguishes between circRNA and homologous linear RNA through proximity effect of TWJ structure and non-specific amplification blocking ability of the three-letter code. Ultimately, this strategy successfully identified circRNA down to 0.1 % abundance. In addition, benefiting from the synergistically accelerated multiple signal amplification in one step, TTSC strategy exhibited good linear relationship of 0.5–1000 pM with a detection limit of 80.6 fM. Notably, the recovery test indicated that the proposed strategy has good prospects for clinical applications. Therefore, TTSC is expected to provide an ideal platform for circRNA detection in biotechnology research and molecular diagnostics.

Dual amplification dynamic DNA network system for CRISPR/Cas12a based p53 gene detection

BackgroundIt is estimated that over 50 % of human cancers are caused by mutations in the p53 gene. Early sensitive and accurate detection of the p53 gene is important for diagnosis of cancers in the early stage. However, conventional detection techniques often suffer from strict reaction conditions, or unsatisfied sensitivity, so we need to develop a new strategy for accurate detection of p53 gene with smart designability, multiple signal amplification in mild reaction conditions. ResultsIn this study, CRISPR/Cas system is exploited in entropy-driven catalysis (EDC) and hybridization chain reaction (CHA) dual signal amplification sensing strategies. The products of both reactions can efficiently and separately activate CRISPR/Cas12a which greatly amplifies the fluorescent signal. The method has good linearity in p53 detection with the concentration ranged from 0.1 fM to 0.5 pM with ultra-low detection limit of 0.096 fM. It also showed good performance in serum, offering potentials for early disease detection. SignificanceThe designed dual amplification dynamic DNA network system exhibits an ultra-sensitive fluorescence biosensing for p53 gene identification. The method is simple to operate and requires only one buffer for the experiment, and meanwhile shows smart designability which could be used for a wide range of markers. Thus, we believe the present work will provide a potential tool for the construction and development of sensitive fluorescent biosensors for diseases.

"Three-in-One": Ultrasensitive Lateral Flow Immunoassay Driven by Magnetic Enrichment and Photothermal Signal Amplification.

Conventional lateral flow immunoassay (LFIA) usually suffers from poor antimatrix interference, unsatisfactory sensitivity, and lack of quantitative ability for target analyte detection in food matrices. In response to these limits, here, multifunctional nanomaterial ZnFe2O4 nanoparticles (ZFOs) were developed and integrated into LFIA for powerful magnetic separation/enrichment and colorimetric/photothermal target sensing. Under optimum conditions, the detection for clenbuterol (CL) with magnetic enrichment achieves 9-fold higher sensitivity compared to that without enrichment and 162-fold higher sensitivity compared to that based on traditional colloidal golds. Attributing the improved performances of ZFOs, CL can be detected at ultralow levels in pork and milk with 10 min of immunoreaction time. The vLODs were 0.01 μg kg-1 for two modes, and the cutoff values of CL were about 5 and 3 μg kg-1, respectively. More importantly, the enrichment ZFO-mediated LFIA (ZE-LFIA) exhibits a similar limit of detection (LOD) in both buffer solution and food matrix, demonstrating a universal resistance to the food matrix. The multitudinous performance merits of this ZE-LFIA with high sensitivity, matrix tolerance, accuracy, and specificity have ensured a broad application potential for target detection of clenbuterol and can serve as an experience for other veterinary drug residues' detection.

A novel sandwich electrochemical immunosensor utilizing customized template and phosphotungstate catalytic amplification for CD44 detection

A sandwich-type electrochemical immunosensor was proposed for the ultra-sensitive detection of CD44, a potential biomarker for breast cancer. In this design, a customized template-based ionic liquid (1-butyl-2,3-dimethylimidazolium tetrafluoroborate) carbon paste electrode (CILE) served as the sensing platform, and thionine/Au nanoparticles/covalent-organic frameworks (THI/Au/COF) were used as the signal label. Moreover, an enzyme-free signal amplification strategy was introduced by involving H2O2 and phosphotungstate (PW12) with peroxidase-like activity. Under optimized conditions, the linear range is as wide as six orders of magnitude, and the detection limit is as low as 0.71 pg mL−1 (estimated based on S/N = 3). Average recoveries range from 98.16 %-100.1 %, with a relative standard deviation (RSD) of 1.42–8.27 % in mouse serum, and from 98.44 %-99.06 %, with an RSD of 1.14–4.84 % (n = 3) in artificial saliva. Furthermore, the immunosensor exhibits excellent specificity toward CD44, good stability, and low cost, indicating great potential for application in clinical trials.

Enzyme-Programmed Self-Assembly of Nanoparticles.

Nanoparticles are a hot topic in the field of nanomaterial research due to their excellent physical and chemical properties. In recent years, DNA-directed nanoparticle self-assembly technology has been widely applied to the development of numerous complex nanoparticle superstructures. Due to the inherent stability and surface electric repulsion of nanoparticles, it is difficult to make nanoparticle superstructures respond to molecular signals in the external environment. In fact, enzyme-programmed molecular systems are developed to allow diverse functions, including logical operations, signal amplification, and dynamic assembly control. Therefore, combining enzyme-controlled DNA systems may endow nanoparticle assembly systems with more flexibility in program design, allowing them to respond to a variety of external signals. In this review, we summarize the basic principles of enzyme-controlled DNA/nanoparticle self-assembly and introduce its applications in heavy metal detection, gene expression, proteins inside living cells, cancer cell therapy, and drug delivery. With the continuous development of new nanoparticle materials and the increasing functionality of enzyme DNA circuits, enzyme-directed DNA/nanoparticle self-assembled probe technology is expected to see significant future development.

High-efficiency electrochemiluminescence of host-guest ligand-assembled gold nanoclusters preoxidated for ultrasensitive biosensing of protein

Herein, host–guest ligand-assembled gold nanoclusters (AuNCs) with highly efficient electrochemiluminescence (ECL) as an emitter and a programable tripedal deoxyribonucleic acid (DNA) walker with a well-regulated DNA track as the signal amplifier were used to develop an ultrasensitive biosensing platform for determining soluble urokinase-type plasminogen activator receptor (suPAR) related to chronic kidney disease. Notably, the host–guest ligand-assembled AuNCs with 6-aza-2-thiothymine (ATT) as the ligand and guanidine butyric acid (Gba) as the model (Gba/ATT-AuNCs) exhibited a strong and stable ECL signal under preoxidation, 25.6 times higher than that of traditional ATT-protected AuNCs (ATT-AuNCs). This is because of the dual-enhancement ECL mechanism of the suppression of nonradiative transitions by host–guest recognition and the improvement of the electrochemical redox rate by preoxidation. Furthermore, the trace target protein suPAR could be efficiently converted into a tripedal DNA walker to walk along a well-regulated square DNA orbit with high movement efficiency and low background signal, thereby considerably improving detection sensitivity. Thus, the established ECL sensing platform achieved the ultrasensitive detection of suPAR with a linear range from 10 fg/mL to 10 ng/mL and a limit of detection of 3.15 fg/mL, whose detection sensitivity was five orders of magnitude higher than that of the gold-standard enzyme-linked immunosorbent assay with the lowest detection concentration of 1 ng/mL. This strategy provides a novel enhanced ECL mode of AuNCs for constructing a sensitive biosensing platform and is expected to be extended to the trace detection of other protein markers in human body fluids for disease diagnosis.

Ultrasensitive Electrochemiluminescence Biosensor Based on DNA-Bio-Bar-Code and Hybridization Chain Reaction Dual Signal Amplification for Exosomes Detection.

Exosomes have received considerable attention as potent reference markers for the diagnosis of various neoplasms due to their close and direct relationship with the proliferation, adhesion, and migration of tumor. The ultrasensitive detection of cancer-derived low-abundance exosomes is imperative, but still a great challenge. Herein, we report an electrochemiluminescence (ECL) biosensor based on the DNA-bio-bar-code and hybridization chain reaction (HCR)-mediated dual signal amplification for the ultrasensitive detection of cancer-derived exosomes. In this system, two types of aptamers were modified on the magnetic nanoprobe (MNPs) and gold nanoparticles (AuNPs) with numerous bio-bar-code DNA, respectively, which formed "sandwich" structures in the presence of specific target exosomes. The "sandwich" structures were separated under magnetic field, and the numerous bio-bar-code DNA were released by dissolving AuNPs. The released bio-bar-code DNA triggered the HCR procedure to produce a good deal of long DNA duplex structure for embedding in hemin, which generated strong ECL signal in the presence of coreactors for ultrasensitive detection of exosomes. Under the optimal conditions, it exhibited a good linearly of exosomes ranging from 10 to 104 exosomes particle μL-1 with limit of detection down to 5.01 exosome particle μL-1. Furthermore, the high ratio of ECL signal and minor change of ECL intensity indicated the good specificity, stability, and repeatability of this ECL biosensor. Given the good performance for exosome analysis, this ultrasensitive ECL biosensor has a promising application in the clinical diagnosis of early cancers.