Signal Amplification Strategy Research Articles

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.

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 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.

A Membrane-Confined Signal Amplification Strategy for Sensitive Monitoring of Extracellular Enzymatic Activity Upon Drug Stimulus.

Extracellular enzymes are not only strongly correlated with disease development but also play critical roles in modulating immune responses. Therefore, real-time monitoring of extracellular enzymatic activity can afford straightforward insights into their spatiotemporal dynamics upon drug stimulus, and provide promising tools to unravel their key roles in modulating the cell signaling. Although DNA-based sensing probes have been frequently developed for the detection of a variety of biomolecules, there still lacks a modular design strategy for amplified imaging of extracellular enzymatic activity associated with live cells. Herein, we developed an enzymatically triggerable signal amplification strategy for real-time dynamic imaging of extracellular enzyme activity through a cell membrane-confined hybrid chain reaction (HCR). We demonstrated that, by modifying the initiator DNA with enzyme-specific incision sites and cholesterol tail, extracellular enzyme-trigged HCR could be fulfilled on the surface of the cellular membrane, facilitating amplified detection of extracellular enzymatic activity. Dynamic monitoring of enzyme secretion of cancer cells upon stimulus or macrophage cells upon inflammation challenge has also been achieved. We envision that the design strategy could provide valuable information for dissecting the role of extracellular enzymes in modulating cell responses to drug treatment.

Novel electrochemiluminescence sensing platform for ultrasensitive detection of malathion residue in tea based on SiO2NSs doped Luminol/AgNPs as a signal amplification strategy

To address the potential hazards of organophosphorus pesticides (OPs) residues in tea, an electrochemiluminescence (ECL) aptasensor based on functionalized nanomaterials was constructed in this work. Firstly, gold nanoparticles (AuNPs) were attached on the surface of multi-walled carbon nanotubes (MWCNTs) by the constant potential electrodeposition to form a compound, and it was utilized to provide excellent immobilization sites for complementary DNA (cDNA). Subsequently, composite nanomaterials were synthesized by a one-pot method with aminated Luminol/silver nanoparticles@silica nanospheres (NH2-Luminol/Ag@SiO2NSs). Finally, NH2-Luminol/Ag@SiO2NSs was combined with a malathion aptamer (Apt) to obtain signal probes (SPs) for the construction of an aptasensor. The aptasensor had a wide linear range (1×10−3-1×103 ng/mL) and a low limit of detection (LOD) (0.3×10−3 ng/mL). It had the virtues of high sensitivity, wonderful stability and excellent specificity, which could be used for the detection of malathion residue in tea. The work provides a proven way for the construction of a rapid and ultrasensitive aptasensor with low-cost.

Dual-enhanced enzyme cascade hybrid hydrogel for the construction of optical biosensor

The biomimetic enzyme cascade system plays a key role in biosensing as a sophisticated signal transduction and amplification strategy. However, constructing a regulated enzyme cascade sensing system remains challenging due to the mismatch of multiple enzyme activities and poor stability. Herein, we design an efficient dual-enhanced enzyme cascade hybrid system (UFD-DEC) containing DNA-controlled nanozymes (Fe-cdDNA) and enzyme (urease) via combining the electrostatic contact effect with the hydrogel-directed confinement effect. Precise modulation of Fe-cdDNA nanozyme by DNA offers a means to control its catalytic efficiency. This regulated UFD-DEC system accelerates the reaction rate and provides remarkable stability compared with the free enzyme system. Benefiting from the plasticity properties of hydrogels, a “lab-in-a-tube” platform was constructed by encapsulating UFD-DEC in a microcentrifuge tube. Such a UFD-DEC-based hydrogel tube exhibits sufficient adaptability to profile urea when used in conjunction with a smartphone-assisted image processing algorithm, which on-site delivers urea information with a detection limit of 0.12 mmol L−1. This customizable and inexpensive miniaturized biosensor platform for monitoring urea may facilitate point-of-care testing applications.

Nanozyme-mediated signal amplification on g-C3N4/NaBiO3 Z-scheme heterojunction photoelectrode toward ultrasensitive photoelectrochemical immunoassay for prostate-specific antigen

A sandwich-type photoelectrochemical (PEC) immunosensor for prostate-specific antigen (PSA) detection was developed using the g-C3N4/NaBiO3 (CN/NBO) Z-scheme heterojunction as the photoelectrode and glutathione-Cu/Cu2O nanozyme (GSH-Cu/Cu2O NPs) as the signal amplifier. The unique Z-scheme charge-carrier migration pathway of the CN/NBO heterojunction significantly promotes the separation and migration of photogenerated electron-hole pairs, producing a high photocurrent response of the photoelectrode. GSH-Cu/Cu2O NPs with ascorbic acid oxidase (AAO) activity were introduced into the PEC immunosensing system through immunoreactions for signal amplification. Signal amplification was achieved through double photocurrent quenching of the GSH-Cu/Cu2O NPs on the photoelectrode. On the one hand, GSH-Cu/Cu2O NPs as AAO catalyzed ascorbic acid (AA), an electron donor in the PEC system, to produce dehydroascorbic acid (DHA), leading to a decrease in the photocurrent response. On the other hand, the GSH-Cu/Cu2O NPs prevented AA transfer from the detection solution to the interface of the photoelectrode, further aggravating the PEC signal quenching effect. Benefiting from the excellent performance of the Z-scheme heterojunction-based photoelectrode, the dual signal quenching effect of GSH-Cu/Cu2O NPs, the PEC immunosensor showed an ultralow detection limit of 5.1 × 10−15 g mL−1. By coupling a Z-scheme heterojunction-based photoelectrode with a nanozyme-mediated signal amplification strategy, this study provides a novel avenue for the construction of ultrasensitive and convenient biosensing systems.

An ultrasensitive fluorescence sensing platform for HCV detection based on the T7 isothermal amplification combined with aggregation-induced emission luminogens strategy

Hepatitis C virus infection constitutes a significant global public health concern. Fluorescence technique (FL) is a promising biosensor, however, many conventional dyes exhibit reduced fluorescence or no emission at high concentrations. To break through this shackle, an ultrasensitive AIE-based (aggregation-induced emission) FL sensing platform was developed for HCV detection by using the FLRNAs-based T7 isothermal transcription amplification RNA diagnosis combined with AIE luminogens (FTAR) strategy. In the FTAR strategy, the T7 transcription amplification process was responsible for the recognition of the target HCV and produced Pepper aptamer, which could form aggregates of nanoparticles with the HBC, an AIEgen, to produce high-intensity FL. The multi-integrated signal amplification strategy led to ultrasensitivity. Moreover, under the optimal experimental conditions, HCV could be assayed in the range of 100aM-100pM. The proposed strategy has been successfully applied in detecting HCV in clinical samples. In summary, this research was the first to utilize aptamer restriction of AIEgens internal rotation to achieve aggregation-induced emission and presented the successful development of a specific, sensitive and rapid FTAR assay for HCV diagnosis. The proposed FTAR platform with facile operation, short analysis time, low-cost as well as excellent quantification ability could provide a promising tool for point-of-care testing (POCT).

Hook-Like DNAzyme-Activated Autocatalytic Biosensor for the Universal Detection of Pathogenic Bacteria.

DNAzyme-based assays have found extensive utility in pathogenic bacteria detection but often suffer from limited sensitivity and specificity. The integration of a signal amplification strategy could address this challenge, while the existing combination methods require extensive modification to accommodate various DNAzymes, limiting the wide-spectrum bacteria detection. We introduced a novel hook-like DNAzyme-activated autocatalytic nucleic acid circuit for universal pathogenic bacteria detection. The hook-like connector DNA was employed to seamlessly integrate the recognition element DNAzyme with the isothermal enzyme-free autocatalytic hybridization chain reaction and catalytic hairpin assembly for robust exponential signal amplification. This innovative autocatalytic circuit substantially amplifies the output signals from the DNAzyme recognition module, effectively overcoming DNAzyme's inherent sensitivity constraints in pathogen identification. The biosensor exhibits a strong linear response within a range of 1.5 × 103 to 3.7 × 107 CFU/mL, achieving a detection limit of 1.3 × 103 CFU/mL. Noted that the sensor's adaptability as a universal detection platform is established by simply modifying the hook-like connector module, enabling the detection of various pathogenic bacteria of considerable public health importance reported by the World Health Organization, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella typhimurium. Additionally, the specificity of DNAzyme in bacterial detection is markedly improved due to the signal amplification process of the autocatalytic circuit. This hook-like DNAzyme-activated autocatalytic platform presents a versatile, sensitive, and specific approach for pathogenic bacteria detection, promising to significantly expand the applications of DNAzyme in bacteria detection.

Novel pore confinement enhanced europium functionalized porphyrin covalent organic framework electrochemiluminescence microreactor and its application to microRNA-21 detection

Here, an electrochemiluminescence (ECL) microreactor lanthanide europium functionalized porphyrin covalent organic framework (Eu@TAPP-COF) was developed through Schiff base condensation between 1,3,5-benzenetricarboxaldehyde (BTCA) and 5,10,15,20-tetrakis(4-aminophenyl)-porphyrin (TAPP) with the coordination reaction between the COF skeleton and Eu3+. Compared with O2/S2O82− and TAPP-COF/S2O82− systems, the ECL intensity of Eu@TAPP-COF/S2O82− is significantly enhanced by 5.1 and 1.3 times, respectively. It can be attributed to the positively charged micropore structure of the Eu@TAPP-COF, providing a relatively independent reaction environment for the electrochemical oxidation of S2O82− and the intermediate SO4•−, moreover shortening the distance among the ECL luminophore TAPP, the co-reaction accelerator Eu3+, and the co-reaction reagent S2O82−. Based on this novel emitter, an ECL biosensor was developed for microRNA-21 detection via the signal amplification strategy of catalytic hairpin assembly (CHA). The as-prepared biosensor displayed high sensitivity for target with a dynamic linear response range from 100 aM to 100 pM, and a lower detection limit of 21 aM (S/N = 3). This work provides an idea for the sensing application of weak luminophore and a simple, effective and accurate detection platform for tumor markers.

SERS biosensors based on catalytic hairpin self-assembly and hybridization chain reaction cascade signal amplification strategies for ultrasensitive microRNA-21 detection.

A highly sensitive surface-enhanced Raman scattering (SERS) biosensor has been developedfor the detection of microRNA-21 (miR-21) using an isothermal enzyme-free cascade amplification method involving catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). The CHA reaction is triggered by the target miR-21, which causes hairpin DNA (C1 and C2) to self-assemble into CHA products. After AgNPs@Capture captures the resulting CHA product, the HCR reaction is started, forming long-stranded DNA on the surface of AgNPs. A strong SERS signal is generated due to the presence of a large amount of the Raman reporter methylene blue (MB) in the vicinity of the SERS "hot spot" on the surface of AgNPs. The monitoring of the SERS signal changes of MB allows for the highly sensitive and specific detection of miR-21. In optimal conditions, the biosensor exhibits a satisfactory linear range and a low detection limit for miR-21 of 42.3 fM. Additionally, this SERS biosensor shows outstanding selectivity and reproducibility. The application of this methodology to clinical blood samples allows for the differentiation of cancer patients from healthy controls. As a result, the CHA-HCR amplification strategy used in this SERS biosensor could be a useful tool for miRNA detection and early cancer screening.

Ultrasensitive self-powered biosensor with facile chemical signal amplification strategy using hydrogen peroxide-triggered silver oxidation reaction

The amplification strategies used for self-powered biosensor based on biofuel cell (BFC-SPB) need to be further developed. Because the currently developed strategies utilized the complicated hybridization of DNA or poorly readable current signal of capacitors for amplification, which limits the practical application in public health emergencies. Here, we present a facile chemical amplification strategy for BFC-SPB. The 5-min amplification was triggered by simply adding H2O2 solution dropwise to the sensing cathode after the formation of the immune sandwich. The Ag NP of immunoprobe were oxidized to Ag(I), which can be served as the electron acceptor of the cathode. The amount of immunoprobe was positively correlated with that of the antigen, resulting in corresponding and high concentration of Ag(I) after the amplification, which enhanced the ability of the cathode as the electron acceptor. Meanwhile the glucose oxidation reaction (GOR) was performed on the bioanode modified with glucose oxidase (GOx). After assembling the bioanode and sensing cathode, the open circuit voltage of the BFC-SPB, measured by digital multimeter, distinctly rised with the elevated concentration of the antigen. To demonstrate the proof of concept, immunoglobulin G (IgG), selecting as a model analyte, was sensitively detected using this method. Result indicated that the limit of detection was 4.4 fg mL−1 (0.03 amol mL−1) in the linear range of 1 pg mL−1-10 μg mL−1. This work initiates a brand-new way of chemical amplification strategy for BFC-SPB, and offers a promising platform for practical applications.

Synergistic powering of DNA walker movement by endogenous dual enzymes for constructing dual-mode biosensors

To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01–500 U mL−1, presenting a good linearity in the concentration range of 0.01–10 U mL−1, with a detection limit of 0.01 U mL−1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01–50 U mL−1 and reducing the detection limit to 0.002 U mL−1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.

Biomarker Multiplexing with Rational Design of Nucleic Acid Probe Complex

AbstractThe expression profiles of intracellular biomarkers hold significance for understanding cellular biological functions and tracking pathological activities. Due to its programmability and biocompatibility, extensive efforts have been devoted to design various kinds of nucleic acid probes for biomarker detection. However, pinpointing a single biomarker could end up in a false positive signal, delaying diagnosis. In this review, we present an overview of current advances in biomarker detection and signal amplification techniques. We highlight strategies for biomarker multiplexing and signal amplification with combination of isothermal approaches. High specificity and sensitivity are the two criteria for a desired probe, as are the challenges encountered by a probe that operates efficiently in biological systems. With higher biomarker identification accuracy, we may be able to move one step closer to precision medicine.

A portability self-powered sensor facilitates sensitive Cd2+ detection: Dual mechanism and three quantitative mode

As a common heavy metal contaminant, Cd2+ has adverse effects on food safety and consumer health. It is very important for human health to realize highly sensitive Cd2+ detection methods. The self-powered sensing system based on enzyme biofuel cells (EBFCs) does not need an external power supply, which can simplify the experimental equipment and has great application value in portable detection. Thus, the biosensor is innovatively integrated into the screen-printed electrode to construct a new type of portable sensor suitable for on-site and real-time Cd2+ detection. Hybridization chain reaction (HCR) combined with the Cd2+-dependent deoxyribose (DNAzyme) signal amplification strategy is used to enhance the detection sensitivity while specifically recognizing the Cd2+. Moreover, the self-powered sensor combines with smartphones to realize quantitative Cd2+ detection without other instruments and has the characteristic of Effectively improving the hazard detection technology is essential to ensure food safety. Portability, simplicity, and speed are suitable for real-time Cd2+ detection in the field. The dual mechanism and three quantitative modes combining colorimetric and two electrical signals output modes are adopted to realize the visualization and accurate detection. A series of research results confirm that this strategy is of great significance to strengthen the development of intelligent Cd2+ technology, expand the application of self-powered sensing technology, and improve the safety detection system.

The sensing of circRNA with tetrahedral DNA nanostructure modified microfluidic chip

BackgroundCircular ribonucleic acids (circRNAs) are a type of covalently closed noncoding RNA with disease-relevant expressions, making them promising biomarkers for diagnosis and prognosis. Accurate quantification of circRNA in biological samples is a necessity for their clinical application. So far, methods developed for detecting circRNAs include northern blotting, reverse transcription quantitative polymerase chain reaction (RT-qPCR), microarray analysis, and RNA sequencing. These methods generally suffer from disadvantages such as large sample consumption, cumbersome process, low selectivity, leading to inaccurate quantification of circRNA. It was thought that the above drawbacks could be eliminated by the construction of a microfluidic sensor. ResultsHerein, for the first time, a microfluidic sensor was constructed for circRNA analysis by using tetrahedral DNA nanostructure (TDN) as the skeleton for recognition probes and target-initiated hybridization chain reaction (HCR) as the signal amplification strategy. In the presence of circRNA, the recognition probe targets the circRNA-specific backsplice junction (BSJ). The captured circRNA then triggers the HCR by reacting with two hairpin species whose ends were labeled with 6-FAM, producing long DNA strands with abundant fluorescent labels. By using circ_0061276 as a model circRNA, this method has proven to be able to detect circRNA of attomolar concentration. It also eliminated the interference of linear RNA counterpart, showing high selectivity towards circRNA. The detection process can be implemented isothermally and does not require expensive complicated instruments. Moreover, this biosensor exhibited good performance in analyzing circRNA targets in total RNA extracted from cancer cells. SignificanceThis represents the first microfluidic system for detection of circRNA. The biosensor showed merits such as ease of use, low-cost, small sample consumption, high sensitivity and specificity, and good reliability in complex biological matrix, providing a facile tool for circRNA analysis and related disease diagnosis in point-of care application scenes.

A Dual-Recognition Fluorescence Enzyme-Linked Immunosorbent Assay for Specific Detection of Intact Lipid Nanoparticles via a Localized Scaffolding Autocatalytic DNA Circuit Amplifier.

Lipid nanoparticles (LNPs) are emerging as one of the most promising drug delivery systems. The long-circulating effect of intact LNPs (i-LNPs) is the key to efficacy and toxicity in vivo. However, the significant challenge is specific and sensitive detection of i-LNPs. Herein, a dual-recognition fluorescence enzyme-linked immunosorbent assay (DR-FELISA) was developed to directly isolate and detect i-LNPs by combining dual-recognition separation with a one-step signal amplification strategy. The microplates captured and enriched i-LNPs through antibody-antigen reaction. Dual-chol probes were spontaneously introduced into the lipid bilayer of captured i-LNPs, converting the detection of i-LNPs into the detection of double-cholesterol probes. Finally, the end of the dual-chol probes initiated the localized scaffolding autocatalytic DNA circuits (SADC) system for further signal amplification. The SADC system provides a sensitive and efficient amplifier through localized network structures and self-assembled triggers. Simultaneous recognition of i-LNPs surface PEG-lipid and lipid bilayer structures significantly eliminates interference from biological samples. i-LNPs were detected with high selectivity, ranging from 0.2 to 1.25 mg/mL with a limit of detection of 0.1 mg/mL. Moreover, this method allows the isolation and quantitative analysis of different formulations of i-LNPs in serum samples with a satisfactory recovery rate ranging from 94.8 to 116.3%. Thus, the DR-FELISA method provides an advanced platform for the exclusive and sensitive detection of i-LNPs, providing new insights for the study of the quality and intracorporal process of complex formulations.

HCR-Assisted RTF-EXPAR-Based Lateral Flow Analysis for Sensitive Detection of H1N1 Influenza Virus.

The H1N1 influenza virus is a significant pathogen responsible for seasonal influenza, and its frequent outbreaks pose substantial challenges to global public health. The present study successfully developed a lateral flow analysis platform that integrates reverse transcription-free exponential amplification reaction (RTF-EXPAR) and hybridization chain reaction (HCR) processes with functionalized quantum dots for the direct detection of H1N1 influenza virus RNA, eliminating the need for reverse transcription. The fluorescence signal on the band recorded with a smartphone can be utilized for the quantitative determination of the target. Interestingly, the dual signal amplification strategy exhibits high sensitivity with a remarkably low detection limit of 10 aM. Moreover, this platform exhibits excellent flexibility and universality, where the various pathogens can be determined by replacing the specific nucleic acid fragments in RTF-EXPAR. The aforementioned advantages reveal its huge potential in the early diagnosis of H1N1 influenza virus infection and developing point-of-care testing (POCT) equipment for nucleic acid analysis.

DSN signal amplification strategy based nanochannels biosensor for the detection of miRNAs

MiRNA-21 is recognized as an important biological marker for the diagnosis, treatment, and prognosis of breast cancer. Here, we have created a nanochannel biosensor utilizing the duplex-specific nuclease (DSN) signal amplification strategy to achieve the detection of miRNAs. In this system, DNA as the capture probe was covalently immobilized on the surface of nanochannels, which hybridized with the target miRNA and forms RNA/DNA duplexes. DSN could cleave the probe DNA in RNA/DNA duplexes, recycling target miRNA, which may again hybridized with other DNA probes. After N cycles, most of the DNA probes had been cleaved, and the content of miRNA could be quantified by detecting changes in surface charge density. This biosensor can distinguish miR-21 from non-complementary miRNAs and one-base mismatched miRNAs, with reliable detection limits as low as 1 fM in PBS. In addition, we had successfully applied this method to analysis of total RNA samples in MCF-7 cells and HeLa cells, and the nanochannels had also shown excellent responsiveness and strong anti-interference ability. This new method is expected to contribute to miRNA detection in clinical diagnostics, providing a unique approach to detecting and distinguishing disease-associated molecules.

Novel dynamic light scattering immunosensor for prostate specific antigens based upon dual-tyramine signal amplification strategy

The sensitive detection of biomarkers is crucial for the early diagnosis, treatment, and improvement of the quality of life of patients. A novel dynamic light scattering-dual tyramine signal amplification strategy (DLS-dTSA) was constructed to detect prostate-specific antigen (PSA). The Oligo 1 was first immobilized in a 96-well plate for PSA capture. Afterwards, horseradish peroxidase (HRP) and Oligo 2 functionalized AuNPs (HRP-Au NPs-Oligo 2) bound to Oligo 1 through DNA hybridization. In the presence of hydrogen peroxide (H2O2), tyramine could be converted into a transient radical intermediate by HRP, leading to the aggregation of tyramine and HRP-labeled AuNPs (tyramine-AuNPs-HRP) near HRP-AuNPs-Oligo 2, triggering the first TSA process. Later, the PSA was added into the 96-well plate, to specifically recognize aptamer and thus depart the Au-aggregate from the 96-well plate. Then H2O2 was added to the Au-aggregate solution, triggering the free Au-aggregate to gather again, realizing the second TSA amplification. The average diameter of Au NPs increased linearly with the concentration of PSA during the range of 0.0001 – 1000 ng mL−1, and the limit of detection was 0.033 pg mL−1. Moreover, this strategy has been used to analyze PSA in human serum samples successfully, demonstrating the potential application of the proposed method for rapid clinical and early clinical diagnosis of prostate cancer in vitro.