Signal Amplification Strategy Research Articles

Simultaneous detection of CaMV35S and NOS using fluorescence sensors with dual-emission silver nanoclusters and catalytic hairpin amplification strategy.

Adual-emission fluorescent biosensing method was developed for simultaneous determinationof CaMV35S and NOS in genetically modified(GM) plants. Two designed hairpin DNA (H1, H2) sequences were used as templates to synthesize H1-AgNCs (λex = 570nm, λem = 625nm) and H2-AgNCs (λex = 470nm, λem = 555nm). By using H1-AgNCs and H2-AgNCs as dual-signal tags, combined with signal amplification strategy of magnetic separation to reduce background signal and an enzyme-free catalytic hairpin assembly (CHA) signal amplification strategy, a novel multi-target fluorescent biosensor was fabricated to detect multiple targets based on FRET between signal tags (donors) and magnetic Fe3O4 modified graphene oxide (Fe3O4@GO, acceptors). In the presence of the target NOS and CaMV35S, the hairpin structures of H1 and H2 can be opened respectively, and the exposed sequences will hybridize with the G-rich hairpin sequences HP1 and HP2 respectively, displacing the target sequences to participate in the next round of CHA cycle. Meanwhile, H1-HP1 and H2-HP2 double-stranded DNA sequences (dsDNA) were formed, resulting in the desorption of dsDNA from the surface of Fe3O4@GO due to weak π-π interaction between dsDNA and Fe3O4@GO and leading to the fluorescence recovery of AgNCs. Under optimal conditions, the linear ranges of this fluorescence sensor were 5 ~ 300nmol L-1 for NOS and 5 ~ 200nmol L-1 CaMV35S, and the LODs were 0.14nmol L-1 and 0.18nmol L-1, respectively. In addition, the fluorescence sensor has good selectivity for the detection of NOS and CaMV35S in GM soybean samples, showing the potential applications in GM screening.

Signal amplification strategy based on target-controlled release of mediator for ultrasensitive self-powered biosensing of acetamiprid

Self-powered biosensors with high sensitivity have garnered significant interest for their potential applications in the realm of portable sensing. Herein, a self-powered biosensor with a novel signal amplification strategy was developed by integrating target-controlled release of mediator with an enzyme biofuel cell for the ultrasensitive detection of acetamiprid (ACE). Zeolitic imidazolate framework-67 was utilized as both a nanocontainer for capturing the electron mediator 2,2′-azidobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and a precursor for the synthesis of cobalt nanoparticles/nitrogen, sulfur-codoped carbon nanotubes (Co NPs/NS-CNTs), which were employed as the electrode material for constructing both the glucose oxidase-based bioanode and the laccase-based biocathode. The target analyte ACE can specifically bind to its aptamer, leading to the release of ABTS, which cyclically participates in the catalytic reaction of the biocathode, thereby amplifying the electrochemical signal. By leveraging the benefits of ABTS cyclic catalysis and the effective electrocatalysis of bioelectrodes based on Co NPs/NS-CNTs, the self-powered biosensor has a broad detection range of 0.1–1000 fM and a low detection limit of 25 aM toward ACE. The proposed signal amplification approach presents a promising strategy for enhancing sensitivity and enabling portable analysis in applications of food safety, environmental monitoring, and medical diagnostics.

Immuno-Rolling Circle Amplification (Immuno-RCA): Biosensing Strategies, Practical Applications, and Future Perspectives.

In the rapidly evolving field of life sciences and biomedicine, detecting low-abundance biomolecules, and ultraweak biosignals presents significant challenges. This has spurred a rapid development of analytical techniques aiming for increased sensitivity and specificity. These advancements, including signal amplification strategies and the integration of biorecognition events, mark a transformative era in bioanalytical precision and accuracy. A prominent method among these innovations is immuno-rolling circle amplification (immuno-RCA) technology, which effectively combines immunoassays with signal amplification via RCA. This process starts when a targeted biomolecule, such as a protein or cell, binds to an immobilized antibody or probe on a substrate. The introduction of a circular DNA template triggers RCA, leading to exponential amplification and significantly enhanced signal intensity, thus the target molecule is detectable and quantifiable even at the single-molecule level. This review provides an overview of the biosensing strategy and extensive practical applications of immuno-RCA in detecting biomarkers. Furthermore, it scrutinizes the limitations inherent to these sensors and sets forth expectations for their future trajectory. This review serves as a valuable reference for advancing immuno-RCA in various domains, such as diagnostics, biomarker discovery, and molecular imaging.

Mg2+- dependent six-jawed trap-mediated SERS biosensor for ratiometric detection of microRNA with use of the highly ordered Au NCs as substrate

Herein, a ratiometric surface-enhanced Raman scattering (SERS) biosensor was constructed with use of the highly ordered gold nanocubes (Au NCs) interface created by the liquid-phase assembly as substrate, and a novel Mg2+-dependent six-Jawed trap (SJT) cyclical amplification strategy for sensitive detection of microRNA 221 (miRNA-221). Impressively, in contrast to conventional gold nanoparticles (Au NPs), Au NCs with more sharp corners and edge characteristics can significantly improve the Raman signals of the Raman molecules, while the highly ordered Au NCs prepared by liquid-phase assembly can also increase the stability and repeatability of miRNA-221 detection. In addition, the newly constructed SJT with more Mg2+ shear sites were utilized to efficiently release plentiful output DNA with the Raman molecule methylene blue (MB) for greatly enhancing the sensitivity of the SERS biosensor. Ultrasensitive ratiometric SERS detection of miRNA-221 was realized by capturing the output DNA containing MB to generate ratiometric Raman signals at the sensing interface modified with Cyanine 5 (Cy5) Raman molecules. The SERS biosensor analyzed for miRNA-221 from 1 fM - 10 nM with an identification limit of 0.66 fM, and the approach is also effective for detecting miRNA-221 in cell lysates. Notably, the SJT signal amplification strategy utilized in this study makes it easier to create extremely sensitive biosensors and opens up new avenues for early detection of hepatocellular carcinoma cells (HCC).

Dually-amplified electrochemical aptasensor based on the self-linking AuPt nanoflowers for ultrasensitive determination of hemagglutinin

BackgroundInfluenza virus can spread from person to person and cause epidemics. Therefore, rapid and sensitive diagnosis of virus is essential in controlling influenza outbreaks. Conventional virus diagnostic techniques are time-consuming, labor-intensive and requires large instruments. In this work, a sandwich electrochemical assay by a pair of aptamers was developed for ultrasensitive determination of hemagglutinin (HA) protein, which is one of the two surface glycoproteins of influenza A (H1N1) virus, using dual signal amplification techniques. ResultsHA was captured and magnetically separated by Fe3O4@SiO2-NH2@Au attached to aptamer 1 (Apt1), creating a sandwich structure with AuPt nanoflowers (AuPtNFs) connected to aptamer 2 (Apt2). Herein, AuPtNFs could catalyze H2O2/hydroquinone (HQ) to generate 1,4-benzoquinone (BQ), and achieved amplification of electrochemical signal detection through differential pulse voltammetry (DPV). The constructed aptasensor expressed a wide linear range (10 pg/mL-100 ng/mL) with limit of detection (LOD) of 2.4 pg/mL. Moreover, a novel strategy for dual signal amplification was developed to further enhance sensitivity. The innovative electrochemical aptasensor could achieve secondary amplification of the detection signal with LOD of 0.3 pg/mL and linear concentration range from 0.5 pg/mL to 100 ng/mL. The secondary amplification could be achieved only through the self-linking process, which allowed for the retention of numerous AuPtNFs by simple complementary base pairing to connect more AuPtNFs onto the above-mentioned sandwich structure. SignificanceOverall, the constructed aptasensor exhibited favorable sensitivity and accuracy, indicating the potential expanded application for the clinical detection of numerous viruses.

Low-cost signal enhanced colorimetric and SERS dual-mode paper sensor for rapid and ultrasensitive screening of mercury ions in tea

Mercury ions (Hg2+) are highly toxic heavy metals that are commonly found in natural environments. Owning to their non-biodegradability and accumulation in the food chain, the precise detection of trace amounts of Hg2+ is essential for preventing chronic accumulation and ensuring food safety. In this study, we present a dual-mode paper sensor for simultaneous colorimetric and Surface-Enhanced Raman Spectroscopy (SERS) detection of Hg2+ in tea, achieving ultrasensitive, rapid, and on-site screening. 4-Mercaptopyridine (4-MPY) was effectively chemisorbed onto the gold nanoparticles (AuNPs), acting as a signal probe for colorimetric methods. Moreover, it can produce plasmonic hot spots for SERS by interacting with the pyridine ring. To enhance the signal intensity of both colorimetry and SERS, a silver shell is in-situ grown on the surface of AuNPs captured on the paper sensor by reduction of Ag+, achieving signal amplification. The visual limit of detection (LOD) for the colorimetric biosensor is 2.5 pM, while the LOD of SERS is 0.48 pM with this dual-mode paper sensor. The sensitivity of both the colorimetric method and SERS was improved by approximately 200 and 500 times, respectively, with the designed signal amplification strategy. The system allows for multiple parallel screening of the same sample, ensuring accurate results without any false-positive or false-negative. This study provides a valuable platform for the accurate detection of various other heavy metal ions and provides effective strategies for improving the performance of colorimetric methods.

A Triple Signal Amplification Strategy for Accurate and Ultrasensitive miRNA-21 Detection.

A triple signal amplification strategy was integrated with a built-in double electrode and external energy storage device to fabricate a novel self-powered biosensor for ultrasensitive detection of miRNA-21. Specifically, DNA tetrahedra and haripin2-glucose oxidase are modified on the surface of the biocathode and bioanode by catalytic hairpin assembly (CHA) to achieve dual signal amplification. Moreover, triple signal amplification is realized by including an external capacitor. Consequently, the as-constructed self-powered biosensor demonstrates a low detection limit of 0.06 fM toward the miRNA-21 assay within the range of 0.1 fM to 10 pM. This study presents a practical and sensitive approach to timely cancer detection.

Fluorescence and photoacoustic (FL/PA) dual-modal probe: Responsive to reactive oxygen species (ROS) for atherosclerotic plaque imaging

Accurate and early detection of atherosclerosis (AS) is imperative for their effective treatment. However, fluorescence probes for efficient diagnosis of AS often encounter insufficient deep tissue penetration, which hinders the reliable assessment of plaque vulnerability. In this work, a reactive oxygen species (ROS) activated near-infrared (NIR) fluorescence and photoacoustic (FL/PA) dual model probe TPA-QO-B is developed by conjugating two chromophores (TPA-QI and O–OH) and ROS-specific group phenylboronic acid ester. The incorporation of ROS-specific group not only induces blue shift in absorbance, but also inhibits the ICT process of TPA-QO-OH, resulting an ignorable initial FL/PA signal. ROS triggers the convertion of TPA-QO-B to TPA-QO-OH, resulting in the concurrent amplification of FL/PA signal. The exceptional selectivity of TPA-QO-B towards ROS makes it effectively distinguish AS mice from the healthy. The NIR emission can achieve a tissue penetration imaging depth of 0.3 cm. Moreover, its PA775 signal possesses the capability to penetrate tissues up to a thickness of 0.8 cm, ensuring deep in vivo imaging of AS model mice in early stage. The ROS-triggered FL/PA dual signal amplification strategy improves the accuracy and addresses the deep tissue penetration problem simultaneously, providing a promising tool for in vivo tracking biomarkers in life science and preclinical applications.

Novel Self-Powered Biosensor Based on Au Nanoparticles @ Pd Nanorings and Catalytic Hairpin Assembly for Ultrasensitive miRNA-21 Assay.

An ultrasensitive self-powered biosensor is constructed for miRNA-21 detection based on Au nanoparticles @ Pd nanorings (Au NPs@Pd NRs) and catalytic hairpin assembly (CHA). The Au NPs@Pd NRs possess excellent electrical conductivity to improve the electron transfer rate and show good elimination of byproduct H2O2 to assist glucose oxidase (GOD) to catalyze glucose; CHA is used as an amplification strategy to effectively enhance the sensitivity of the biosensor. To further amplify the output signal, a capacitor is integrated into the self-powered biosensor. With multiple signal amplification strategies, the self-powered biosensor possesses a linear range of 0.1-10-4 fM and a lower limit of detection (LOD) of 0.032 fM (S/N = 3). In addition, the as-prepared self-powered biosensor displays potential applicability in the assay toward miRNA-21 in human serum samples.

Near-infrared light-enhanced colorimetric signal amplification strategy for tumor marker detection based on MoS2/CuO/Au nanocomposite.

Anovel signal amplification strategy was developedby combining near-infrared light with MoS2/CuO/Au nanocomposite for building acolorimetric immunoassay. First, MoS2/CuO/Au nanocomposite was synthesized by precipitation and photoreduction methods and characterized by scanning electron microscopy (SEM) and X-ray powder diffraction (XRD). MoS2/CuO/Au nanocomposite has oxidase-like activity and can oxidize TMB to form a blue product (TMBox). Further, the catalytic oxidation of TMB was accelerated under near-infrared (NIR) laser radiation. The sandwich-type colorimetric immunoassay was constructed using MoS2/CuO/Au nanocomposite. Under the enhancement of near-infrared light, carcinoembryonic antigen (CEA) was sensitively detected in the range 0.1 to 40ng/mL with the limit of detection of 0.03ng/mL. Moreover, the immunosensor has excellent selectivity and anti-interference, good repeatability, and stability.

Recent Advances in DNA-Based Nanoprobes for In vivo MiRNA Imaging.

As a post transcriptional regulator of gene expression, miRNA is closely related to many major human diseases, especially cancer. Therefore, its precise detection is very important for disease diagnosis and treatment. With the advancement of fluorescent dye and imaging technology, the focus has shifted from in vitro microRNAs (miRNA) detection to in vivo miRNA imaging. This concept review summarizes signal amplification strategies including DNAzyme catalytic reaction, hybrid chain reaction (HCR), catalytic hairpin assembly (CHA) to enhance detection signal of lowly expressed miRNAs; external stimuli of ultraviolet (UV) light or near-infrared region (NIR) light, and internal stimuli such as adenosine triphosphate (ATP), glutathione (GSH), protease and cell membrane protein to prevent nonspecific activation for the avoidance of false positive signal; and the development of fluorescent probes with emission in NIR for in vivo miRNA imaging; as well as rare earth nanoparticle based the second near-infrared window (NIR-II) nanoprobes with excellent tissue penetration and depth for in vivo miRNA imaging. The concept review also indicated current challenges for in vivo miRNA imaging including the dynamic monitoring of miRNA expression change and simultaneous in vivo imaging of multiple miRNAs.

Quantum-Dot-Encoded Beads-Enhanced CRISPR/Cas-Based Lateral-Flow Assay for the Amplification-Free, Sensitive, and Rapid Detection of Nucleic Acids in Breast Cancer.

Nucleic acid detection plays a pivotal role in the accurate diagnosis of diseases. The CRISPR/Cas detection system, noted for its significant utility in a variety of applications, often necessitates enhanced sensitivity or specific signal amplification strategies, particularly for detecting low-abundance biomarkers. In this study, we present a quantum-dot-encoded beads (QDB)-energized CRISPR/Cas12-based lateral-flow assay (QDB-CRISPR-LFA). This method enables amplification-free, sensitive, and rapid detection (<40 min) of BRCA-1. We validated our method using contrived reference samples and nucleic acids extracted from tumor cells. The QDB-CRISPR-LFA provides a visual, more rapid alternative to the traditional BRCA-1 real-time RT-PCR assay. Significantly, through the integration of CRISPR's specificity and the high signal output of QDB, the detection threshold for BRCA-1 has been reduced to the femtomolar level, representing an enhancement of 2-4 orders of magnitude over existing CRISPR/Cas detection methods. This advancement underscores the potential of our approach in advancing nucleic acid detection techniques, which is crucial for the early and precise diagnosis of diseases.

A sandwich−type photoelectrochemical sensor constructed with a signal amplification strategy based on the upconversion luminescence characteristics of Ag@N−CQDs for sensitive detection of neuron specific enolase

In this paper, Bi2S3/AgBiS2 composite nanomaterials and PDA@Ag@N−CQDs were synthesized, and used as substrates and second antibody label respectively to construct a sandwich photoelectrochemical (PEC) sensor. The upconversion luminescence effect of N−CQDs can convert long wavelength light into short wavelength light that can be utilized by the substrate material, which can provide additional excitation light energy for the substrate material and further enhance the photoelectric response. Besides, Ag has SPR effect and can also promote electron transfer. The proposed sandwich immunosensor achieves detection of NSE in the concentration range of 0.001 ng mL−1 to 100 ng mL−1, with a detection limit of 0.28 pg mL−1 (S/N = 3). What's more, the proposed sensor also exhibits good stability, selectivity, as well as reproducibility, indicating its promising application prospects.

A quantitative method for aquaporin-1 protein using magnetic preconcentration and probe-based immunoassay coupling to inductively coupled plasma mass spectrometry in urine analysis

BackgroundAquaporin-1 (AQP1) protein plays a crucial role in intracellular and extracellular water homeostasis and fluid transport in organs and tissues associated with diverse life activities and is extremely abundant in the kidney. Accurate detection of AQP1 in urine can be applied as screening of early-stage disease. Application of magnetic preconcentration and probe-based signal amplification strategy coupling to inductively coupled plasma mass spectrometry (ICP-MS) is a more accurate, sensitive and specific detection method for AQP1 in complex biological samples compared to conventional methods. ResultsWe described an element-labelling strategy based on magnetic preconcentration and probe-based immunoassay coupling to ICP-MS detection. The magnetic beads (MBs) modified with epoxy groups were capable of enriching AQP1 proteins and separating them from complex matrices. The probe constructed by conjugating anti-AQP1 antibody molecules on the surface of gold nanoparticles could specifically recognize AQP1 proteins attached on MBs and be analyzed by ICP-MS. The concentration of AQP1 protein could be precisely quantified and amplified by 14,000 times through the corresponding signal of Au atoms. This assay for AQP1 protein quantification achieved a detection limit down to 0.023 ng mL−1, a broad linear calibration curve between 0.3 ng mL−1 and 30 ng mL−1, as well as outstanding specificity. SignificanceThe proposed method was successfully applied to detect AQP1 protein in human urine samples, showing the potential for its applications concerning accurate AQP1 quantification. It can also screen a wide range of proteins provided the antibodies specific to these target proteins are available.

Fluorescent and colorimetric dual-readout platform for tuberculosis point-of-care detection based on dual signal amplification strategy and quantum dot nanoprobe

Rapid and accurate diagnosis of tuberculosis (TB) is of great significance to control the spread of this devastating infectious disease. In this work, a sensitive and low-cost point-of-care testing (POCT) detection platform for TB was developed based on recombinase polymerase amplification (RPA)-catalytic hairpin assembly (CHA)-assisted dual signal amplification strategy. This platform could achieve homogeneous fluorescent and visual diagnosis of TB by using CdTe quantum dots (QDs) signal reporter. In the presence of target DNA (IS1081 gene fragment), RPA amplicons blocked by short oligonucleotide strands could trigger CHA signal amplification, leading to the Ag+ releasing from C–Ag+-C structure and the fluorescence quenching of CdTe QDs by the released Ag+. Furthermore, the detection performance of CdTe QDs modified by 3-mercaptopropionic acid (MPA) or thiomalic acid (TMA) (MPA-capped QDs and TMA-capped QDs) was systematically compared. Experimental results demonstrated that TMA-capped QDs exhibited better detection sensitivity due to their stronger interaction with Ag+. The limits of detection (LODs) of fluorescence and visual analysis were as low as 0.13 amol L−1 and 0.33 amol L−1. This method was successfully applied to the clinical sputum samples from 36 TB patients and 20 healthy individuals, and its quantitative results were highly consistent with those obtained by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The proposed approach has the advantages of high sensitivity and specificity, simple operation and low cost, and is expected to be applied in clinical TB screening and diagnosis.

Enzyme-free fluorescent DNA detection based on nucleic acid-templated click reaction via controllable synthesis of Cu2O as heterogeneous nanocatalyst

In the field of nucleic acid amplification assays, developing enzyme-free, easy-to-use, and highly sensitive amplification approaches remains a challenge. In this work, we synthesized a heterogeneous Cu2O nanocatalyst (hnCu2O) with different particle sizes and shapes, which was used for developing enzyme- and label-free nucleic acid amplification methods based on the nucleic acid-templated azide–alkyne cycloaddition (AAC) reaction catalyzed by hnCu2O. The hnCu2O exhibited size- and shape-dependent catalytic activity, with smaller sizes and spherical-like shapes exhibiting superior activity. Spherical-like hnCu2O (61 ± 8 nm) not only achieved a ligation yield of up to 84.2 ± 3.9 % in 3 min but also exhibited faster kinetics in the nucleic acid-templated hnCu2O-catalyzed AAC reaction, with a high reaction rate of 0.65 min−1 and a half-life of 1.07 ± 0.09 min. Based on this result, we developed nucleic acid-templated click ligation linear amplification reaction (NA-CLLAR) and nucleic acid-templated click ligation exponential amplification reaction (NA-CLEAR) approach. By combining the recognition (complementary to the target sequence) and signal output (split G-quadruplex sequence) elements into a DNA probe, the NA-CLLAR and NA-CLEAR fluorescence assays achieved highly specific detection of target nucleic acids, with a detection limit of 2.8 aM based on G-quadruplex-enhanced fluorescence. This work is a valuable reference and will inspire researchers to design enzyme-free nucleic acid signal amplification strategies by developing different types of Cu(I) catalysts with improved catalytic activity.

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.