Signal Amplification Strategy Research Articles

Quenched Electrochemical Biosensor Based on Adsorption Between GO and Aptamer for the Detection of ERα

Breast cancer is one of the three most common cancers in the world, and is the most prevalent malignancy in women. Estrogen receptor alpha (ERα), an important marker for early diagnosis of breast cancer, shows positivity in more than 75% of breast cancers. Herein, a quenched electrochemical biosensor based on the adsorption interaction between graphene oxide (GO) and aptamer (Apt) and the ATRP signal amplification strategy for sensitive detection of ERα was constructed. The Apt was immobilized on the electrode surface by gold-sulfur bonding and adsorbed GO-BIBB by π–π stacking. In the ATRP reaction solution, ferrocene polymerized on the electrode surface in the presence of a catalyst and initiator and generated electrochemical signal. After the addition of ERα, a large number of electrical signal molecules were shed from the electrode surface with GO, causing quenching of the signal. By optimizing the reaction conditions, the detection limit (LOD) of the biosensor can reach 0.17 pg·ml−1, and the logarithm of the current intensity and concentration shows a good linearity in the range of 1 pg·ml−1 to 100 ng·ml−1. The biosensor also has excellent selectivity, stability, interference resistance and reproducibility. More importantly, the biosensor enabled the detection of ERα in real samples.

Signal Amplification Strategy Design in Nanozyme‐Based Biosensors for Highly Sensitive Detection of Trace Biomarkers (Small Methods 11/2023)

Signal Amplification Strategy Design in Nanozyme‐Based Biosensors for Highly Sensitive Detection of Trace Biomarkers (Small Methods 11/2023)

Recent Advances in Biological Applications of Aptamer-Based Fluorescent Biosensors.

Aptamers have been spotlighted as promising bio-recognition elements because they can be tailored to specific target molecules, bind to targets with a high affinity and specificity, and are easy to chemically synthesize and introduce functional groups to. In particular, fluorescent aptasensors are widely used in biological applications to diagnose diseases as well as prevent diseases by detecting cancer cells, viruses, and various biomarkers including nucleic acids and proteins as well as biotoxins and bacteria from food because they have the advantages of a high sensitivity, selectivity, rapidity, a simple detection process, and a low price. We introduce screening methods for isolating aptamers with q high specificity and summarize the sequences and affinities of the aptamers in a table. This review focuses on aptamer-based fluorescence detection sensors for biological applications, from fluorescent probes to mechanisms of action and signal amplification strategies.

A point-of-care solid-phase colorimetric sensor based on the enzyme-induced metallization for ALP detection

Alkaline phosphatase (ALP) is a crucial biomarker for clinical diagnosis, which is closely related to the physiological homeostasis regulation process of human body. And the abnormal level of ALP is associated with numerous diseases, such as liver dysfunction, bone diseases, diabetes, and so on. In order to meet the demand of personalized healthcare, it is particularly important to develop a miniaturized point-of-care testing (POCT) device for ALP detection. Herein, a portable solid-phase colorimetric sensor based on enzyme-induced metallization signal amplification strategy was constructed for ALP detection. The AuNPs modified on the glass slides acted as crystal seeds, allowing Ag+ in the solution to be reduced and deposited on the surface of AuNPs, which further formed the gold core and silver shell (Au@Ag) complex and generated visual signals. The visual signals were recorded by a smartphone and quantified using open-source ImageJ software. Under the optimal conditions, the proposed method exhibited a good linear relationship from 2.0 to 16.0 pM, and the detection limit was as low as 0.9 pM. In addition, it was further successfully applied for ALP detection in non-transparent and complex samples (milk, different types of cells). A sensitive, low cost, rapid and convenient solid-phase sensor was developed for ALP detection, which was expected to provide a promising strategy for POCT devices.

Chemical-chemical redox cycling for improving the sensitivity of the fluorescent assay: A proof-of-concept towards DNA methylation detection

Ultrasensitive analytical methods are still urgent for the discovery of trace level biomarkers and the early clinical diagnosis of disease. In this work, an ultrasensitive universal sensing platform was constructed by integrating fluorescent assay with chemical-chemical redox cycling signal amplification strategy. Using Ru@SiO2 nanoparticles wrapped by MnO2 nanosheets (Ru@SiO2@MnO2) as fluorescent probe, the chemical-chemical redox cycling system was conducted upon ascorbic acid (AA) and tris(2-carboxyethyl)phosphine (TCEP) as reductants and MnO2 nanosheets as oxidant. The MnO2 nanosheets not only could quench the fluorescence of Ru@SiO2 nanoparticles to reduce the background, but also could serve as oxidants to react with AA, generating dehydroascorbic acid (DHA). The DHA was reduced by TCEP in turn to form AA that participated in the next cycling of chemical-chemical redox reaction. Thus, the constantly released AA from the chemical-chemical redox cycling system could massively etch MnO2 nanosheets on Ru@SiO2 surface, making the fluorescence of Ru@SiO2 nanoparticles greatly recovered. It was shown that the sensitivity of the fluorescent assay was improved almost 52 times by utilizing the chemical-chemical redox cycling signal amplification strategy. This strategy was further employed to detect DNA methylation with the aid of AA-encapsulated liposomes that were modified with 5 mC antibodies to bind with the methylated DNA captured in 96-well plate. A detection of limit down to 16.2 fM was achieved for the detection of methylated DNA. It's believed that the incorporation of chemical-chemical redox cycling signal amplification strategy into fluorescent sensing paves a new way for ultrasensitive detection of biomarkers.

Photocurrent-polarity switching between methylene blue-loaded liposome and iodine-doped BiOCl for in-situ amplified immunoassay

This work designed a liposome-mediated photocurrent polarity switching immunosensor depending on the reversed photocurrent of iodine-doped BiOCl (I-BOC) nanoflowers induced by the released methylene blue (MB) for the detection of prostate-specific antigen (PSA). Initially, MB-loaded liposomes as indicators were confined within the microplates to participate in the sandwiched immunoreaction and lysed under the treatment of Triton X-100 to release numerous MB. Owing to the host-guest recognition between β-cyclodextrin (β-CD) and MB, the released MB was immobilized on the β-CD-modified I-BOC/FTO electrode and triggered the photocurrent polarity reversal from cathodic photocurrent to anodic photocurrent. The sensing platform realized an accurate and sensitive assay of PSA due to the effective elimination of false-positive/negative signals in a linear range of 0.02–50 ng mL−1 with a limit of detection of 12 pg mL−1. Furthermore, this work not only conjugated liposome-assisted signal amplification strategy with the photocurrent polarity switching system but also provided a novel pathway for various protein determinations.

Signal amplification strategy by chitosan-catechol hydrogel modified paper electrode for electrochemical detection of trace arsenite

Arsenic contamination is widespread in water bodies and poses a significant risk to human health, thus developing a rapid and inexpensive in situ method for the detection of arsenic is urgently needed. In this study, a chitosan-catechol carbon paper electrode was fabricated by electrodeposition of chitosan on a conductive carbon paper and subsequential electro-oxidative grafting of catechol. This chitosan-catechol modified electrode demonstrated good performance in detecting trace amounts of arsenic, which is mainly due to the significantly amplification of oxidation signals generated by ferrocene (Fc) on chitosan-catechol hydrogel. The interaction of As(III) with quinone and semiquinone radicals results in the decrease of electronic transmission nodes. The modified electrode exhibited a good detection capability of low concentration As(III) with a detection limit 0.82 ppb by differential pulse voltammetry (DPV). In addition, the chitosan-catechol modified sensor has the merits of high reproducibility, durability and strong resistance to ionic interference. Further successful detection of As(III) in actual rice and soil samples suggests its good potential in the field of in situ detection.

Effects of High-Biotin Sample Interference on Antibody Concentrations in Sandwich Immunoassays.

The use of antimicrobial growth promoters (AGPs) is banned because of problems associated with drug residues in animal products and increased bacterial resistance. The immunization of chickens with specific antigens is a promising strategy for generating specific antibodies that can target a wide range of antibiotic-resistant bacteria and can be used as an alternative to antibiotics. Immunoglobulin Y (IgY) antibodies in a polyclonal antibody (pAb) format, when administered orally, modulate the ruminal microbiome and maintain animal health and performance; however, there are concerns pertaining to protein impurities and biotin concentrations in the samples. Signal amplification strategies involving the noncovalent interaction of biotin with streptavidin is extensively used in diagnosis and scientific research, particularly in enzyme-linked immunosorbent assays (ELISAs). However, the high concentrations of biotin in samples, especially in those derived from rich sources such as egg yolk, can pose challenges and potentially harm the accuracy of diagnostic tests and protein concentration measurements. This study aimed to evaluate the influence of biotin on the measurement of IgY in freeze-dried egg yolk samples obtained from immunized laying hens using immunoassays with biotin-avidin/streptavidin. The detection of IgY in yolk samples using ELISA with streptavidin-biotin binding could lead to misdiagnosis due to biotin interference; the level of interference varies with the specific assay conditions and the concentration of biotin in the yolk samples. An ELISA without streptavidin-biotin binding is advisable to avoid interactions between biotin and target proteins, prevent biotin interference with the results, and achieve more reliable and accurate results.

Enhanced magnetic relaxation switching immunoassay for chlorpyrifos based on tyramine signal amplification

AbstractChlorpyrifos, an organophosphorus pesticide, is widely used in agriculture to protect crops from insects. However, the presence of its residues in food has caused widespread concern due to the serious risks to human health. Traditional detection methods suffer from limitations, such as low sensitivity, long detection time, and complicated operations. Herein, based on the tyramine signal amplification (TSA) strategy, we developed a sensitive and rapid magnetic relaxation switching (MRS) immunosensor for the detection of chlorpyrifos. Wherein, magnetic Fe3O4 nanoparticles modified with tyramine (MNP150‐tyramine) acted as magnetic probes for magnetic relaxation signal output. In the presence of hydrogen peroxide (H2O2) and horseradish peroxidase (HRP), tyramine can be converted to the highly reactive intermediate that covalently binds with the nearby proteins, such as HRP and antibody, thus assembling MNPs to magnetic clusters and showing changes in transverse relaxation time (T2) signals. Based on the “antibody–antigen” immunoreaction, chlorpyrifos could make a connection of HRP/antibody‐modified MNPs and MNP150‐tyramine with a result of MNPs aggregation and strong T2 signals. In this study, the TSA‐MRS method showed sensitive detection of chlorpyrifos with a limit of detection of 0.54 ng/mL, a 4.5‐fold enhancement in the sensitivity compared with the ELISA method, providing an alternative method for the detection of harmful substances in food samples.

Highly sensitive detection for xanthine by combining single-band red up-conversion nanoparticles and cycle signal amplification strategy based on internal filtration effect

Up-conversion nanoparticles (UCNPs), especially single-band bright red UCNPs, have better penetration of biological tissues, absorb less lost energy, and have higher sensitivity and accuracy in the determination of actual biological samples in the field of biosensing. Here, a novel colorimetric and fluorescent dual-channel method based upon an internal filtration effect (IFE) quenching mechanism was proposed for the quantitative analysis of xanthine (XA) by using red UCNPs as fluorescence indicator and 3,3′,5,5′ -tetramethylbenzidine (TMB) as chromogenic substrate. The sensitivity of the detection system was also enhanced by a cycle signal amplification strategy based on the Fenton reaction. Under the best conditions, the detection limits of XA by fluorescent and colorimetric methods were 0.58 μM and 1.19 μM, respectively. The developed method was applied to the detection of XA in actual serum samples, and the recoveries of the spiked samples by fluorescent and colorimetric methods were in the range of 96.3–104.3 % and 94.3–105.4 %, respectively. In addition, the commercial ELISA method was used to verify the application of the proposed method and the test results of XA were close to those obtained by fluorescent and colorimetric methods, indicating that the accuracy of the developed nanosensing system was acceptable.

A novel ternary Y-DNA walker amplification strategy designed fluorescence aptasensor based on Au@SiO2@Fe3O4 nanomaterials for ochratoxin A detection.

Anovel ternary Y-DNA walker amplification strategy designed fluorescence aptasensor based on Au@SiO2@Fe3O4 nanomaterials for ultrasensitive and specific ochratoxin A detection in food samplesis presented. Au@SiO2@Fe3O4 nanomaterials provide the loading platform as well as separation and recovery properties for the ternary Y-DNA walker. The ternary Y-DNA walker is designed to be driven by Nb.BbvCI cleavinga large number of FAM probes to achieve signal amplification. SinceOchratoxin A (OTA) can bind to the constituent aptamer in the ternary Y-DNA walker, adding OTA will destroy the structure of the ternary Y-DNA walker, thereby inhibiting the driving process of the walker. After optimization of various parameters, a standard curve was obtained from 100 to 0.05ng·mL-1 of OTA with the limit of determination of0.027ng·mL-1. The spiked recovery of peanut samples by this method was 82.00-93.30%, and the aptasensorshowed excellent specificity and long-term stability. This simple, robust, and scalable oligonucleotide chain-based ternary Y-DNA walker can provide a general signal amplification strategy for trace analysis.

A smartphone-assisted electrochemiluminescent detection of miRNA-21 in situ using Ru(bpy)32+@MOF

We have proposed a signal dual-amplification electrochemiluminescence (ECL) strategy based on tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) as chromophores confined with three-dimensional (3D) zinc oxalate metal−organic frameworks (Ru(bpy)32+@MOFs) for the detection of miRNA-21. The three-dimensional chromophore connectivity in zinc oxalate MOFs provided a network among Ru(bpy)32+ units, shielding the chromophores from solvent molecules and resulting in high Ru(II) complex emission efficiency. Additionally, we discovered that magnetic beads (MBs) used as carrier for enriched signals contribute to enhanced ECL intensity of the chromophore. To evaluate its clinical application, we applied this method to determine the concentration of miRNA-21 solutions ranging from 1.56 to 100 nM, obtaining a calibration curve of ECL intensity versus logarithm of concentration (logC) of miRNA-21 with a high correlation coefficient. This work demonstrates the construction of a signal amplification strategy ECL biosensor for miRNA using Ru(bpy)32+@MOF systems and its application in ECL detection for analyte methodology.

Mxene quantum dots bipolar electrochemiluminescent platform for hepatitis C virus envelope protein E2 detection

A burgeoning diversified closed bipolar electrochemiluminescent (d-BPE-ECL) based on photothermal amplification biosensor via the thermophysical and photochemical properties of niobium carbide Mxene quantum dots (Nb2C MQDs) has been proposed. The device consists of three components: separated intermediate recognition, cathodic catalytic hydrogen evolution reaction (HER) and anodic ECL response channel. Wherein, the recognition compartment was innovatively designed as a temperature-sensitive conductivity modulated interface, and the introduction of photothermal material PDA@Nb2C MQDs through target mediated rolling circle amplification strategy increases the interface temperature under near-infrared light radiation, thereby enhancing the BPE current and leading to the amplification of the anode ECL signal of Nb2C MQDs. In addition, MoS2@Ni–Cu–P features excellent electrocatalytic activity, which can promote HER and thus accelerate electron transfer, further amplifying the ECL signal. Therefore, a highly sensitive d-BPE-ECL biosensor for hepatitis C virus envelope protein E2 detection with a linear range from 10−4 to 10 ng/mL and detection limit of 3.3 × 10−5 ng/mL was obtained. This work is expected to provide a new direction for exploring BPE multiple signal amplification strategy and broaden the application of BPE-ECL in bioassays.

An accurate and sensitive sensor based on the internal filtration effect for the detection of alkaline phosphatase and ascorbic acid by the Fenton reaction and dual readout signal

Ascorbic acid (AA) and alkaline phosphatase (ALP) in human serum are indispensable substances in the process of biological metabolism, and their abnormal contents are strongly associated with a range of diseases. Therefore, it is meaningful to develop a simple, accurate and sensitive assay for the detection of AA and ALP. Up-conversion nanoparticles (UCNPs) have some unique characteristics, especially, the potential for background-free excitation by the near-infrared region (NIR) significantly improves the limits of detection (LODs). Moreover, sensors based on the inner filter effect (IFE) do not require covalent linkage between receptors and fluorophores or surface modification, thus the probe fabrication is simple and flexible UCNP-based. Therefore, a simple, selective, sensitive, and flexible sensing system for the detection of AA and ALP is proposed by combining the IFE and the cyclic signal amplification strategy. Under ideal circumstances, the LODs of ALP through the colorimetric and fluorescence method were 0.192 U/L and 0.013 U/L. The established sensing platform was applied to assessing ALP activity in human serum, and the detection results are close to those of commercially available kits, indicating its immense potential as an effective and convenient tool for monitoring ALP activity in clinical settings.

Laccase-inspired bi-amino acid MOFs with high substrate affinity: Catalytic deposition induced “signal-down” electrochemical response towards PD-L1

A dual-ligand Cu-MOFs (Cu-F/Hs) based on phenylalanine (Phe) and histidine (His) was innovatively constructed with enhanced catalytic property. Among them, Phe can interact with the dopamine (DA) to enhance the substrate affinity. Meanwhile, the coordination of His with copper ions was inspired by the active site structure of natural laccase to improve the catalytic activity. The Km value of Cu-F/Hs was 0.14 times of laccase, indicating a high affinity for the catalytic substrate DA. Accordingly, a sensitive sensor for PD-L1 detection was designed by using Cu-F/Hs, which can catalyze DA to form redox-active polymeric conjugates deposited on the electrode surface with good electrical conductivity, thereby contributing a “signal-down” electrochemical response with a double signal enhancement. The detection limit was 0.12 ng/mL in the detection range of 0.5–200 ng/mL. This work provided a new and simple way to synthesize MOFs with high substrate affinity and efficient activity, and a general signal amplification strategy induced by the catalytic deposition.

Nitrogen-Doped Graphene Quantum Dots as Electrochemiluminescence-Emitting Species for Sensitive Detection of KRAS G12C Mutation via PET-RAFT.

The levels of KRAS G12C point mutation is recognized to be closely related to the earlier diagnosis of non-small cell lung cancer (NSCLC). Here, based on nitrogen-doped graphene quantum dots (NGQDs) and photo-induced electron/energy transfer reversible addition-fragment chain transfer (PET-RAFT) signal amplification strategy, we fabricated a novel electrochemiluminescence (ECL) biosensor for the detection of KRAS G12C mutation for the first time. NGQDs as ECL-emitting species with cathodic ECL were prepared by a simple calcination method. Firstly, KRAS G12C mutation DNA, i. e., target DNA (tDNA), was captured by specific identification with hairpin DNA (hDNA). Then, PET-RAFT was initiated by blue light, and large numbers of monomers were successfully polymerized to form controllable polymer chains. Lastly, massive NGQDs was introduced via amidation reaction with N-(3-aminopropyl)methacrylamide hydrochloride (APMA), which significantly amplified the ECL signal intensity. Under optimal conditions, this biosensor achieved a good linear relationship between ECL intensity and logarithm of the levels of KRAS G12C mutation in the range from 10 fM to 10 nM. Moreover, this strategy exhibited high selectivity and excellent applicability for KRAS G12C mutation detection in the serum samples. Therefore, this biosensor has great potential in clinical diagnosis and practical application.

Ultrasound-enhanced catalytic hairpin assembly capable of ultrasensitive microRNA biosensing for the early screening of Alzheimer's disease

Catalytic hairpin assembly (CHA) is a promising enzyme-free, isothermal signal amplification strategy, but the relatively time-consuming strand replacement limits its application scenarios. Here, we developed an ultrasound-enhanced catalytic hairpin assembly (UECHA) biosensing platform for early screening of Alzheimer's disease by introducing a portable acoustic-drive platform with functionalized microspheres for effective biomarkers enrichment and fluorescence enhancement. By constructing a gradient ultrasonic field in a microcavity, the platform concentrates the functionalized microspheres in a central position, accompanied by an enhanced fluorescence signal with a specific release. In addition, the programmable frequency modulation can also modify the acoustic potential well and effectively promote non-equilibrium chemical reactions such as CHA (25 min). Compared with the conventional catalytic hairpin assembly (CHA), UECHA allows for direct and quantitative measurement of AD miRNAs down to 3.55 × 10−15 M in 1 μL samples. This visual analysis of ultra-trace biomarkers based on acoustic enrichment and promotion provides a new perspective for the rapid and highly sensitive clinical detection of Alzheimer's disease.

Signal Amplification Strategy Design in Nanozyme-Based Biosensors for Highly Sensitive Detection of Trace Biomarkers.

Nanozymes show great promise in enhancing disease biomarker sensing by leveraging their physicochemical properties and enzymatic activities. These qualities facilitate signal amplification and matrix effects reduction, thus boosting biomarker sensing performance. In this review, recent studies from the last five years, concentrating on disease biomarker detection improvement through nanozyme-based biosensing are examined. This enhancement primarily involves the modulations of the size, morphology, doping, modification, electromagnetic mechanisms, electron conduction efficiency, and surface plasmon resonance effects of nanozymes for increased sensitivity. In addition, a comprehensive description of the synthesis and tuning strategies employed for nanozymes has been provided. This includes a detailed elucidation oftheir catalytic mechanisms in alignment with the fundamental principles of enhanced sensing technology, accompanied by the presentation of quantitatively analyzed results. Moreover, the diverse applications of nanozymes in strip sensing, colorimetric sensing, electrochemical sensing, and surface-enhanced Raman scattering have been outlined. Additionally, the limitations, challenges, and corresponding recommendations concerning the application of nanozymes in biosensing have been summarized. Furthermore, insights have been offered into the future development and outlook of nanozymes for biosensing. This review aims to serve not only as a reference for enhancing the sensitivity of nanozyme-based biosensors but also as a catalyst for exploring nanozyme properties and their broader applications in biosensing.

Cascade signal amplification electrochemical biosensor based on AgNPs and ring opening polymerization for determination of Ochratoxin A.

Anochratoxin A (OTA) electrochemical biosensor based on a cascade signal amplification strategy with Ag nanoparticles (AgNPs) and ring opening polymerization (ROP) was constructed. The large specific surface area of AgNPs was used to increase the loading of OTA aptamer on the electrode surface, enhancing the ability to capture OTA as a way to achieve the first signal amplification. The OTA antibody modified with polyethylenimine specifically recognizes the OTA, forming an aptamer-OTA-antibody sandwich structure. The amino group on polyethylenimine initiates the ROP reaction with α-amino acid-n-carboxylic anhydride-ferrocene (NCA-Fc) as the monomer. A large number of electrochemical signal units of ferrocene are introduced into the sensing system for a second signal amplification. By amplifying the signal twice, the sensitivity of the sensor is improved. Under the optimal conditions, the detection range of the sensor is 1pg·mL-1 ~ 1μg·mL-1, while the detection limit is as low as 117fg·mL-1. Moreover, the sensor has the advantages of high sensitivity, good stability and selectivity. Standard addition recovery experiment proved that the sensing system can be successfully used for the detection of OTA in four actual samples with recoveries in the range90.0 to 113% with RSDs of 0.6 to 5.2%, providing a new idea for the pollution assessment of mycotoxins.

A Cascade Signal Amplification Strategy for the Ultrasensitive Fluorescence Detection of Cu2+ via λ-Exonuclease-Assisted Target Recycling with Mismatched Catalytic Hairpin Assembly.

Herein, an ultrasensitive DNAzyme-based fluorescence biosensor for detecting Cu2+ was designed using the cascade signal amplification strategy, coupling λ-exonuclease-assisted target recycling and mismatched catalytic hairpin assembly (MCHA). In the designed detection system, the target, Cu2+, can activate the Cu2+-dependent DNAzyme to cause a cleavage reaction, releasing ssDNA (tDNA). Then, tDNA binds to hairpin DNA (H0) with an overhanging 5'-phosphorylated terminus to form dsDNA with a blunt 5'-phosphorylated terminus, which activates the dsDNA to be digested by λ-Exo and releases tDNA along with another ssDNA (iDNA). Subsequently, the iDNA initiates MCHA, which can restore the fluorescence of carboxyfluorescein (FAM) previously quenched by tetramethylrhodamine (TAMRA), resulting in a strong fluorescent signal. Furthermore, MCHA efficiently improves the signal-to-noise ratio of the detection system. More importantly, tDNA recycling can be achieved with the λ-Exo digestion reaction to release more iDNA, efficiently amplifying the fluorescent signal and further improving the sensitivity to Cu2+ with a detection limit of 60 fM. The practical application of the developed biosensor was also demonstrated by detecting Cu2+ in real samples, proving it to be an excellent analytical strategy for the ultrasensitive quantification of heavy metal ions in environmental water sources.