Mediated Signal Amplification Research Articles

Dual-OR Logic-Gated Lateral Flow Strip Assay Based on Colorimetric-Fluorescence Dual Indication for Screening of HPV16/18 in Multiple Scenarios.

The incidence of cervical cancer continues to rise in underdeveloped regions due to low human papillomavirus (HPV) vaccination rates and inadequate screening systems. To achieve convenient, rapid, and accurate detection of HPV, we developed a three-wire lateral flow strip assay system based on dual-OR logic gates for rapid and simultaneous detection of HPV subtypes 16 and 18 in a single test. The system combines three-branch-catalytic hairpin assembly (TCHA)-mediated signal amplification with simple OR logic gate-based signal output to improve detection rates while enabling HPV 16/18 subtype identification. The detection limit of the method was calculated to be 10 aM for the selected target sequences. Meanwhile, the method showed excellent specificity with no false-positive output in real-world detection. The sensitivity of the colorimetric test strips exceeded 90%, while the sensitivity of the fluorescence-based test strips surpassed 95% in detecting clinical samples, demonstrating a high degree of concordance with the results obtained from the real-time quantitative polymerase chain reaction (qPCR). This method provides a simple and easy method for the rapid screening of HPV.

Ruthenium based metal–organic frameworks as annihilation electrochemiluminescence emitters coupled with DNA walker mediated signal amplification for sensitive detection of aflatoxin B1

Ruthenium based metal–organic frameworks as annihilation electrochemiluminescence emitters coupled with DNA walker mediated signal amplification for sensitive detection of aflatoxin B1

Multienzymatic Orthogonal Activation of DNA Codec Enables Tumor-Specific Imaging of Base Excision Repair Activity.

Accurate monitoring of base excision repair (BER) activity in cancer cells is critical for advancing the comprehension of DNA repair processes, gaining insights into cancer development, and guiding treatment strategies. However, current assay techniques for assessing BER activity in cancer cells face challenges due to the heterogeneous origins and diversity of BER enzymes. In this work, we present a highly reliable triple loop-interlocked DNA codec (GATED) that enables precise assessment of BER activity in cancer cells through signal amplification mediated by multienzyme orthogonal activation. The GATED device features a dumbbell-shaped DNA probe to encode two BER enzymes for BER-related signal conversion as well as two bound circular DNA to decode the apurinic/apyrimidinic sites for apurinic/apyrimidinic endonuclease 1 (APE1)-mediated signal amplification. Importantly, GATED is orthogonally activated by multiple target BER enzymes (i.e., uracil DNA glycosylase, thymine DNA glycosylase, and APE1), resulting in a unified fluorescent signal that significantly improves the detection specificity and sensitivity to BER enzymes. Additionally, we demonstrate that the GATED has exceptional biostability within complex biological systems, where it was successfully employed to monitor BER activity in cancer cells with high specificity and enabled cell-based high-throughput screening for BER inhibitors. The GATED provides a much-needed tool for the real-time monitoring of BER activity and the screening of BER inhibitors in cancer cells, potentially advancing both the investigation and clinical application of BER biology.

“Off-on” signal-switchable electrochemiluminescence aptasensor based on Cu2O-ABEI-AgNPs mediated signal amplification for the detection of profenofos

An “off-on” signal-switchable electrochemiluminescence (ECL) aptasensor was designed for the detection of profenofos based on Cu2O-ABEI-AgNPs as ECL enhancers. Cubic Cu2O was prepared as a core, and AgNO3 was reduced to silver nanoparticles (AgNPs) by N-(4-Aminobutyl)-N-ethylisoluminol (ABEI) as a shell on the Cu2O surface. The Cu2O with a large surface area could enhance the fixation of AgNPs. Cu2O and AgNPs were utilized as catalysts to enhance the double amplification ECL signals. Additionally, a molecular probe consisting of gold nanoparticles (AuNPs), a T-type aptamer (consisting of thymines “T”, referred to as TApt), and aptamer (Apt) was prepared (referred to as AuNPs-Apt-TApt). The presence of mercury ion (Hg2+) could potentially quench the ECL signal and bind the thymine-Hg2+-thymine (T-Hg2+-T) structure. The structure formed a probe with Apt and AuNPs. The complementary short chain of aptamer (CApt) was designed and immobilized on AgNPs. In the absence of profenofos, the Apt and CApt were bound through complementary base pairing. The probe was immobilized on the electrode, resulting in quenching of the signal (switched into “off”). However, upon the presence of profenofos, the probe was detached from the electrode, and the signal was restored (switched into “on”). Finally, the aptasensor had a broad detection range (1 ×10-3 ng/mL∼1 ×102 ng/mL) with a lower limit of detection (LOD, 3 ×10-4 ng/mL) and an excellent recovery rate (94.34%∼113.80%) for the detection of profenofos in vegetables. The aptasensor had good stability, repeatability and specificity.

Single primer/self-template-powered isothermal amplification for single-molecule quantification of multiple DNA glycosylases

DNA glycosylase assumes a pivotal role in the preservation of genome integrity by recognizing and excising damaged bases. Herein, we develop a single primer/self-template-powered cascade rolling circle amplification (RCA) and apurinic/apyrimidinic endonuclease (APE1)-mediated signal amplification for simultaneous detection of human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG). The uniqueness of this approach lies in the use of custom-designed hairpin probes to establish a prerequisite for RCA: the conformational change of hairpin probe to circular template, which effectively suppress undesired non-specific amplification. We design two hairpin probes featuring elongated stem structure and 3′-phosphate (3′-PO4) modifications. The stems of hairpin probes are modified with glycosylase substrates and BER process can cleave hairpin probes, resulting in the generation of single-stranded DNAs (ssDNAs) with 3′-hydroxyl (3′-OH) ends. These ssDNAs can function as padlock probes to form circular template upon ligation by ligase. Ultimately, the self-template-initiated RCA and APE1-mediated signal amplification cascade occurs, generating a substantial number of free Cy3 and Cy5 fluorophores that can be simply visualized by single-molecule fluorescence detection. This method demonstrates excellent specificity and sensitivity, with a limit of detection (LOD) of 3.42 × 10-12 U/μL for hAAG and 2.41 × 10-12 U/μL for UDG. This method also can be used for screening of potential inhibitor and kinetic parameter measurement. Most importantly, this method enables accurate profiling of glycosylases expression levels in various cancer cell lines and detection of glycosylase activities at the single-cell level, offering a bright future in cancer detection, medical research, and precision medicine.

A label-free and ultrasensitive electrochemical biosensor using hybrid polypyrrole/gold nanoelectrocatalyst mediated signal amplification for the detection of miRNA-21.

The quantitative detection of microRNAs (miRNAs) is crucial for the diagnosis of cancers, while the traditional methods involve complicated procedures and restricted signal gain. In this study, we have established an ultrasensitive electrochemical biosensor by combining a target-induced hybridization reaction and signal amplification strategy for the detection of miRNA-21. The signal amplification is achieved through employing double-stranded DNA as scaffolds for methylene blue (MB) and using a polypyrrole@gold nanocomposite (ppy@AuNPs) as the electrochemical catalyst for further enhancing the signal. Therefore, this proposed electrochemical platform displayed an analytical performance with a wide linear range from 10 fM to 100 nM and a low detection limit down to 5.4 fM. The excellent selectivity allows the biosensor to discriminate miRNA-21 from other miRNAs, even the one base-mismatched sequence. Moreover, this nanoelectrocatalyst-based platform exhibited good reproducibility and remarkable storage stability, which shows great potential for miRNA-21 detection.

Acid-responsive mediated entropy driven signal amplification nanosystem for sensitive imaging of APE1 activity in living cells

Sensitive imaging of intracellular apurinic/apyrimidinic endonuclease 1 (APE1) activity is helpful to understand its physiological function and pathological role. Herein, an acid-responsive mediated entropy-driven signal amplification all-in-one nanosystem was proposed for sensitive imaging of APE1 activity in living cells. The nanosystem was constructed by modifying recognition probe, entropy-driven reaction (EDR) substrate and acid-responsive components on gold nanoparticles (AuNPs). After the nanosystem was endocytosed into the cell, the triplex in the acid-responsive components folded in the lysosome to free its complementary fuel strand. In the presence of APE1, it specifically cleaved the apurinic/apyrimidinic (AP) site in the recognition probe and released the EDR trigger. With the action of EDR trigger, EDR substrate and fuel strand, the EDR was completed and accompanied by the separation of fluorophore and quenching group in EDR substrate, resulting in obvious fluorescence signal recovery. The nanosystem successfully achieved sensitive imaging of APE1 activity in human cervical cancer cells (Hela cells), human breast cancer cells (MCF-7 cells) and human normal colonic epithelial cells (NCM460 cells) and monitored the changes of APE1 activity in Hela cells. The nanosystem provides a reliable approach for intracellular APE1 analysis and related biomedical research.

Sensitive detection of cancer biomarker with enzyme-free mediated cascade signal amplification empowered undisturbed dual-mode assay

Cancer is a serious threat to human health, and early identification of cancer biomarkers is crucial for timely treatment. A dual-mode biosensing platform has been developed to identify colon carcinoma suppressor microRNA-199a (miRNA-199a). A biofuel cell is firstly constructed using hybridization chain reaction and catalytic hairpin assembly self-assembly. The anode is a flexible carbon cloth (CP) loaded with glucose oxidase-functionalized molybdenum disulfide nanorods (MoS2 nanorods), and the bio-cathode is made up of double-stranded deoxyribonucleotide chains generated by nucleic acid amplification technology. The linear range of the electrochemical method is 0.1 fM-100 pM, and the limit of detection is 24.1 aM (S/N = 3). The colorimetric method has a linear range of 0.1 fM-10000 pM with detection limit of 34.5 aM (S/N = 3). The combination of MoS2 nanorods, enzyme-free cascade signal amplification technology, and dual-mode detection strategy provides high sensitivity and accuracy, offering a promising method for accurate determination of cancer biomarkers.

Quantification of Multiplex miRNAs by Mass Spectrometry with Duplex-Specific Nuclease-Mediated Amplification.

Early quantification of multiplex biomarkers such as microRNAs (miRNAs) is critical during disease pathologic development and therapy. To tackle challenges of low abundance and multiplexing, we herein report a mass-encoded biosensing approach with duplex-specific nuclease (DSN) mediated signal amplification. Magnetic Fe3O4 cores are coated with small gold nanoparticles (AuNPs), which are applied to achieve facile DNA immobilization subsequent separation. This biosensor integrates multiple mass reporters corresponding to different targets (five miRNAs as examples). Due to the excellent resolution of mass spectrometry, these targets can be successfully distinguished in a single spectrum. Wide detection ranges from 10 fM to 1 nM are achieved, and the limits of detection are estimated to be 10 fM. High selectivity is promised due to the enzyme activity of DSN, and practical application in human serum samples performs satisfactorily. The number of targets to be tested can be further expanded by designing different specific mass tags in theory. Therefore, the proposed method can be utilized as an important and valuable tool to quantify multiplex miRNAs for disease screening as well as biomedical investigations.

Smartphone-based chemiluminescence detection of aflatoxin B1 via labelled and label-free dual sensing systems

To develop a sensing platform for onsite determination of AFB1 in foodstuffs, we developed smartphone-based chemiluminescence detection of AFB1 via labelled and label-free dual modes. The labelled mode was characteristic of double streptavidin–biotin mediated signal amplification, obtaining limit of detection (LOD) of 0.04 ng/mL in the linear range of 1–100 ng/mL. To reduce the complexity in the labelled system, a label-free mode based on both split aptamer and split DNAzyme was fabricated. A satisfactory LOD of 0.33 ng/mL was generated in the linear range of 1–100 ng/mL. Both labelled and label-free sensing systems achieved outstanding recovery rate in AFB1-spiked maize and peanut kernel samples. Finally, two systems were successfully integrated into smartphone-based portable device based on custom-made components and android application, achieving comparable AFB1 detection ability to a commercial microplate reader. Our systems hold huge potential for AFB1 onsite detection in food supply chain.

Cartridge voltage-sensitive micropump immunosensor based on a self-assembled polydopamine coating mediated signal amplification strategy

Current biosensing detection assays were developed to focus on rapid, low-cost, stable detection for clinical diagnosis and food safety monitoring. In this work, a novel portable cartridge voltage-sensitive micropump immunosensor (CVMS) biosensing device based on the integration of the microchannel circuit biosensing principle and polydopamine (PDA) was presented for rapid and sensitive detection of pathogenic factors in real samples at trace levels. The CVMS can sensitively evaluate voltage signal changes caused by clogging effects in the closed-loop circuit when the insulated microspheres pass through the microchannel. The targets could trigger the immune reaction between antibody-antigens that leads to the change in the concentration of horseradish peroxidase (HRP). And the HRP was further employed to catalyze the polymerization of dopamine into PDA, resulting in the rapid formation of the magnetic @PDA nanoparticles (MNP@PDA) with core-shell structures. The abundant functional groups on the MNP@PDA surface can efficiently adsorb polystyrene microspheres, thus causing changes in the number of polystyrene microspheres (PS). The CVMS can accurately monitor the change of PS with a portable device, which weighs less than 0.8 kg and costs only $50. The completion of CVMS takes 90 min to complete. The limit of detection of this immunosensor for procalcitonin and ochratoxin A were 42 pg/mL and 77 pg/mL, respectively, which improved about 15 folds and 38 folds, respectively, than those of enzyme-linked immunosorbent assay.

Sensitive detection of fat mass and obesity-associated protein based on terminal deoxynucleotidyl transferase-mediated signal amplification

Fat mass and obesity-associated protein (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) motif in ssDNA or RNA, which plays a crucial role in maintaining the relative stability of m6A modification. Meanwhile, dysregulation of FTO expression can lead to cancers and cardiovascular diseases. So quantitative analysis of FTO is of great significance for the research related with its biological functions and application in disease diagnosis. Herein, we propose a highly sensitive biosensor for FTO detection based on terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification. To be specific, FTO can specifically demethylate the substrate probe, and the emerging double strands can be cleaved by DpnII which is a methylation-sensitive restriction endonuclease. The polymerase TdT in turn recognizes the free 3′OH and initiates polymerization to produce a mass of G-quadruplexes. Thioflavin T (ThT) can then be embedded into G-quadruplexes, producing a strong fluorescence. Experimental results manifest that the fluorescence intensity is positively correlated with the abundance of FTO. Moreover, this biosensor also has good specificity and anti-interference, and the limit of detection (LOD) is down to 30.9 fM. Therefore, our biosensor can provide an efficient strategy for FTO analysis without labeling and separation, showing great potential in clinical application.

Contamination-free V-shaped ultrafast reaction cascade transferase signal amplification driven CRISPR/Cas12a magnetic relaxation switching biosensor for bacteria detection

Foodborne pathogenic bacteria seriously endanger human health and must be rapidly identified for control. Magnetic relaxation switching biosensors (MRS) are ideal for rapid bacteria detection due to their high signal-to-noise ratio and immunity to sample matrix signal interference. However, conventional MRS still has some challenges in terms of sensitivity, specificity, and stability due to insufficient cross-linking or non-specific binding of magnetic nanoparticles (MNPs) to the target. To address these challenges, we firstly proposed a novel contamination-free uracil-DNA glycosylase (UDG) assisted V-shaped PCR driven CRISPR/Cas12a-MRS (UPC-MRS) biosensor, which combines contamination-free ultrafast nucleic acid amplification and powerful CRISPR/Cas12a system. It has an extremely specific quadruple signal guarantee realized by the merits of UDG anti-contamination, PCR primer specificity matching, the CRISPR/Cas12a system's precise recognition abilities, and magnetic probe signal unaffected by the sample matrix. As a cascade combined with original terminal deoxynucleotidyl transferase (Tdt)-mediated signal amplification technology, this platform can achieve Salmonella detection at concentrations as low as 53 CFU/mL, which is more sensitive than most existing MRS sensors, and it displays accuracy and applicability in real sample detection. This novel UPC-MRS biosensors avoid the common aerosol pollution problem of previous CRISPR/Cas12a systems which after combining with nucleic acid amplification, hence not only offers an alternative toolbox for Salmonella and other pathogen detection with satisfactory specificity and sensitivity, but also has potential for future applications across diverse fields.

Detection of DNA Methyltransferase Activity via Fluorescence Resonance Energy Transfer and Exonuclease-Mediated Target Recycling.

In this study, a sensitive method for detecting DNA methyltransferase (MTase) activity was developed by combining the effective fluorescence resonance energy transfer (FRET) of cationic conjugated polymers and exonuclease (Exo) III–mediated signal amplification. DNA adenine MTase targets the GATC sequence within a substrate and converts the adenine in this sequence into N6-methyladenine. In the method developed in this study, the methylated substrate is cleaved using Dpn I, whereby a single-stranded oligodeoxynucleotide (oligo) is released. Afterward, the oligo is hybridized to the 3ʹ protruding end of the F-DNA probe to form a double-stranded DNA, which is then digested by Exo III. Subsequently, due to weak electrostatic interactions, only a weak FRET signal is observed. The introduction of the Exo-III–mediated target-recycling reaction improved the sensitivity for detecting MTase. This detection method was found to be sensitive for MTase detection, with the lowest detection limit of 0.045 U/mL, and was also suitable for MTase-inhibitor screening, whereby such inhibitors can be identified for disease treatment.

An electrochemical biosensor for PD-L1 positive exosomes based on ultra-thin two-dimensional covalent organic framework nanosheets coupled with CRISPR-Cas12a mediated signal amplification

Programmed death-ligand 1 protein positive (PD-L1+) exosome is one of the most prospective biomarkers for the diagnosis of cancer. However, the current methods for the detection of PD-L1+ exosomes are usually laborious, time-consuming and require numerous samples. Herein, we report in this paper an electrochemical biosensor for PD-L1+ exosomes detection based on ultra-thin two-dimensional covalent organic framework nanosheets (2D COF NSs) coupled with clustered regularly interspaced short palindromic repeats (CRISPR) Cas 12a for signal amplification. Firstly, COF-367 NSs functionalized with gold nanoparticles (AuNPs) have been synthesized for the modification of the working electrode, thus the proposed sensor can show highly improved electrochemical performance. Secondly, by utilizing the trans-cleavage activity of Cas12a, this sensor can achieve very high sensitivity for the detection of PD-L1+ exosomes. With such design, the biosensor can have a linear range from 1.2 × 102 to 1.2 × 107 particles/μL with a detection limit of 38 particles/μL. Moreover, this sensor can be employed to analyze non-small cell lung cancer (NSCLC) patient serum samples, and successfully identify NSCLC patients, demonstrating the great potential in clinical applications.

A competitive colorimetric aptasensor for simple and sensitive detection of kanamycin based on terminal deoxynucleotidyl transferase-mediated signal amplification strategy

We developed a rapid and sensitive colorimetric biosensor based on competitive recognition between kanamycin (KAN), magnetic beads-kanamycin (MBs-KAN) and aptamer and terminal deoxynucleotidyl transferase (TdT)-mediated signal amplification strategy. In the absence of KAN, aptamers recognize MBs-KAN. TdT can amplify oligonucleotides to the 3′-OH ends of aptamers, with biotin-dUTP being embedded in the long single stranded DNA (ssDNA). Then the assay produced visual readout due to the horseradish peroxidase (HRP)-catalyzed color change of the substrate after the linkage between biotin and streptavidin (SA)-HRP. In the presence of KAN, however, aptamers tend to bind free KAN rather than MBs-KAN. In this case, aptamers are isolated by magnetic separation, resulting in the failure of signal amplification and catalytic signals. This competitive colorimetric sensor showed excellent selectivity toward KAN with the limit of detection (LOD) as low as 9 pM. And recovery values were between 93.8 and 107.8% when spiked KAN in milk and honey samples.

An Electrochemical Biosensor for Pd-L1 Positive Exosomes Based on Ultra-Thin Two-Dimensional Covalent Organic Framework Nanosheets Coupled with Crispr-Cas12a Mediated Signal Amplification

An Electrochemical Biosensor for Pd-L1 Positive Exosomes Based on Ultra-Thin Two-Dimensional Covalent Organic Framework Nanosheets Coupled with Crispr-Cas12a Mediated Signal Amplification

A highly sensitive aptasensor for the detection of prostate specific antigen based on dumbbell hybridization chain reaction

Accurate and sensitive monitoring of disease biomarkers is of remarkable significance for early clinical diagnosis. Prostate specific antigen (PSA) is regarded as a promising tumor biomarker secreted from the prostate epithelial cells. In this study, a highly sensitive electrochemical biosensor for PSA is proposed with split aptamer based recognition and dumbbell hybridization chain reaction (DHCR) mediated signal amplification. The form of DHCR provides great prospect of DNA self-assembly at solid interface with better performances than traditional HCR. Under optimal conditions, the limit of detection for the analysis of PSA is estimated to be 0.5 pg mL−1. In addition, this method performs satisfactorily in clinical serum samples and affords the advantages of high selectivity, stability, as well as good reproducibility. Therefore, it possesses great application potential in the field of clinical diagnosis.

Light scattering intensity as signal transducer to enhance the performance of immunoassay for Cronobacter detection in powdered infant formula

Herein, we propose the light scattering intensity of silver and gold alloy as signal transducer to enhance the sensitivity and dynamic linearity of immunoassay for quantitative detection of Cronobacter muytjensii (C. muytjensii) in powered infant formula (PIF). Silver and gold alloy, namely, silver-coated gold nanocomposite (AuNF@Ag), was obtained by growing silver on the surface of multi-dendritic colloidal gold. The silver growth was triggered by the hydrolysis of urease to urea in the presence of silver nitrate and glucose. Owing to integrating the advantages of light scattering of AuNF@Ag and enzyme-catalyzed mediated signal amplification, the developed light scattering based immunoassay shows high sensitivity for detection of C. muytjensii, with a detection limit of 51 CFU/mL, which is three orders of magnitude lower than that of conventional enzyme-linked immunosorbent assay (ELISA, 6.39 × 104 CFU/mL). In addition, the proposed method also exhibits wide dynamic curve from 3.4 × 102 CFU/mL to 3.4 × 109 CFU/mL for quantitative detection of C. muytjensii, and is significantly superior conventional ELISA (3.4 × 105 CFU/mL to 3.4 × 109 CFU/mL). The accuracy and precision of the proposed immunoassy were evaluated by analysis of C. muytjensii-fortified PIF samples. The average recoveries of the intra- and inter-assays are within 87.90 %–107.91 %, and the coefficient of variation ranges from 2.72 %–9.32 %. This finding indicates an acceptable accuracy for quantitative detection of C. muytjensii in real PIF samples.

Iodide/metal-organic frameworks (MOF) -mediated signal amplification strategy for the colorimetric detection of H2O2, Cr2O72− and H2S

The analytical methods based on colorimetric detection of various analytes have attracted intensive interest. However, most of them display relatively low sensitivity. Herein, a novel colorimetric strategy based on iodide/metal-organic frameworks (MOF)-mediated amplification was developed for low-cost, naked-eye detection and quantification of H2O2,Cr2O72−, and H2S. Cu-MOFs could catalyze the oxidation of the colorless peroxidase substrate TMB to produce a blue product. The published researches mainly focused on the immobilization or integration of a macromolecule, such as natural enzymes, to enhance MOFs catalytic abilities. The use of small molecules to improve the catalytic performance of MOFs has rarely reported. Due to the negligible steric hindrance, iodide could easily be adsorbed in the framework pore of MOFs to conduct the synergic catalytic effect, and shows a high catalytic effect. As a result, the catalytic activity of Cu-MOFs was dramatically enhanced, and thus, the nanocatalyst could act as an amplifier system for target detection. The detection limits obtained by the amplified method are 25, 30, and 0.2 nM, respectively, which are about 200-fold lower than that of the unamplified colorimetric assays. The colorimetric strategy developed herein provides a novel system for the detection of low concentrations of analytes in complex biological samples.