Ultrasensitive and selective DNA detection by hydroxylamine assisted gold nanoparticle amplification

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization which can achieve ultra-sensitivity up to approximately 30 zmol, i.e. 300 aM.     

Ultrasensitive DNA detection based on Au nanoparticles and isothermal circular double-assisted electrochemiluminescence signal amplification

We report here an ultrasensitive DNA detection approach which combines Au NPs enhanced electrochemiluminescence (ECL) of the CdS nanocrystal (NC) film with isothermal circular amplification reaction of polymerase and nicking endonuclease (NEase). By the double-signal amplification, this approach could sensitively respond down to 5 aM DNA.     

Ultrasensitive amperometric aptasensor for the epithelial cell adhesion molecule by using target-driven toehold-mediated DNA recycling amplification

An amperometric aptasensor is reported for the electrochemical determination of the epithelial cell adhesion molecule (EpCAM). It is based on a combination of EpCAM-driven toehold-mediated DNA recycling amplification, the specific recognition of EpCAM aptamer, and its binding to EpCAM. Hairpin probe 1 (Hp1) with a toehold region was modified with a 5′-thiol group (5’-SH) and self-assembled onto the surface of a gold electrode. Upon addition of EpCAM, the probe A (a 15-mer) is liberated from the aptamer/probe A complex and then hybridizes with the toehold domain of Hp1. This results in the exposure of another toehold for further hybridizing with hairpin probe 2 (Hp2) to displace probe A in the presence of Hp2 that was labeled with the electrochemical probe Methylene Blue (MB). Subsequently, liberated probe A is hybridized again with another Hp1 to start the next round of DNA recycling amplification by reusing probe A. This leads to the formation of plenty of MB-labeled DNA strands on the electrode surface and generates an amplified current. This 1:N probe-response amplification results in ultrasensitive and specific detection of EpCAM, with a 20 pg·mL^?1 detection limit. The electrode is highly stable and regenerable. It was successfully applied to the determination of EpCAM in spiked human serum, urine and saliva, and thus provides a promising tool for early clinical diagnosis.

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Graphical abstract Schematic illustration of the electrochemical detection for EpCAM. The method is based on aptamer-based recognition and EpCAM-driven toehold-mediated DNA recycling amplification. Hp1: Hairpin probe 1; Hp2: Hairpin probe 2; MB: Methylene blue; MCH: 6-Mercapto-1-hexanol; EpCAM: Epithelial cell adhesion molecule.

     

Sequence-specific recognition of HIV-1 DNA based upon nicking-assisted strand displacement amplification and triplex DNA

Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus(HIV) infection diagnosis.Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification(SDA) and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeficiency syndrome(AIDS) in the future.     

Electronic transduction of DNA sensing processes on surfaces: amplification of DNA detection and analysis of single-base mismatches by tagged liposomes.

Tagged, negatively charged, liposomes are used to amplify DNA sensing processes. The analyses of the target DNA are transduced electrochemically by using Faradaic impedance spectroscopy, or by microgravimetric measurements with Au-quartz crystals. By one method, a probe oligonucleotide (1) is assembled on Au-electrodes or Au-quartz crystals. The formation of the double-stranded assembly with the analyte DNA (2) is amplified by the association of the 3-oligonucleotide-functionalized liposomes to the sensing interface. The target DNA is analyzed by this method with a sensitivity limit that corresponds to 1 x 10(-12) M. A second method to amplify the sensing of the analyte involves the interaction of the 1-functionalized electrode or Au-quartz crystal with the target DNA sample (2) that is pretreated with the biotinylated oligonucleotide (4). The formation of the three-component double-stranded assembly between 1/2/4 is amplified by the association of avidin and biotin-labeled liposomes to the sensing interfaces. By the secondary association of avidin and biotin-tagged liposomes, a dendritic-type amplification of the analysis of the DNA is accomplished. The analyte DNA (2) is sensed by this method with a sensitivity limit corresponding to 1 x 10(-13) M. The biotin-tagged liposomes are also used to probe and amplify single-base mismatches in an analyte DNA. The 6-oligonucleotide-functionalized Au-electrode or Au-quartz crystal was used to differentiate the single-base mismatch (G) in the mutant (5) from the normal A-containing gene (5a). Polymerase-induced coupling of the biotinylated-C-base to the double-stranded assembly generated between 6 and 5 followed by the association of avidin and biotin-tagged liposomes is used to probe the single base mismatch. The functionalized liposomes provide a particulate building unit for the dendritic amplification of DNA sensing.     

Photoelectrochemical biosensing platform for the SARS-CoV-2 spike and nucleocapsid proteins

The COVID-19 pandemic is still a continuing worldwide challenge for public health systems. Early and ultrasensitive identification of the infection is essential for preventing the spread of COVID-19 by pre-symptomatic or asymptomatic individuals, particularly in the community and in-home settings. This work presents a versatile photoelectrochemical (PEC) immunosensor for SARS-CoV-2 detection based on a composite material formed by bismuth vanadate (BiVO₄) and strontium titanate (SrTiO₃). The PEC platform was denoted as BiVO₄/SrTiO₃/FTO, and it can be tuned for the detection of either Spike (S) or Nucleocapsid (N) protein by simply altering the antibody immobilized on the platform's surface. Chemical, morphological, and electrochemical characterizations were performed by X-Ray Diffraction, Scanning Electron microscopy, Energy-dispersive X-ray spectroscopy, Electrochemical Impedance Spectroscopy, and Amperometry. With a simple sensing architecture of the PEC platform, it was possible to achieve a linear response range of 0.1 pg mL⁻¹ to 1000 ng mL⁻¹ for S protein and 0.01 pg mL⁻¹ to 1000 ng mL⁻¹ for N protein. The PEC immunosensors presented recovery values for the two SARS-CoV-2 proteins in artificial saliva samples between 97 % and 107.20 % suggesting a good accuracy for the proposed immunosensors.     

Ultrasensitive electrochemical immunoassay based on counting single magnetic nanobead by a combination of nanobead amplification and enzyme amplification

An ultrasensitive electrochemical immunoassay method based on counting single magnetic nanobead (MNB) with combined amplification of nanobead and enzyme. Carcinoembryonic antigen (CEA) and MNB were initially immobilized on substrate at 1:1 molar ratio through sandwich immunoreactions. The MNBs were then labeled with alkaline phosphatase (AP) and continuously introduced to capillary with disodium phenyl phosphate (DPP). APs convert DPPs into a phenol zone around each moving MNB, i.e., one CEA. The phenol zones can be electrochemically detected as peaks and counted for CEA quantification. The detection limit for CEA is 5.0 × 10^?17 mol/L.     

Ultrasensitive and selective detection of nicotinamide adenine dinucleotide by target-triggered ligation-rolling circle amplification

An ultrasensitive fluorescence assay for nicotinamide adenine dinucleotide (NAD(+)) was developed by target-triggered ligation-rolling circle amplification (L-RCA). This novel approach can detect as low as 1 pM NAD(+), much lower than those of previously reported biosensors, and exhibits high discrimination ability even against 200 times excess of NAD(+) analogs.     

Polyphosphonate induced coacervation of chitosan: Encapsulation of proteins/enzymes and their biosensing

Based on the polyphosphonate-assisted coacervation of chitosan, a simple and versatile procedure for the encapsulation of proteins/enzymes in chitosan–carbon nanotubes (CNTs) composites matrix was developed. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), energy dispersive spectrum (EDS) mapping demonstrated the hemoglobin (Hb) uniformly distributed into chitosan–CNTs composites matrix. Raman measurements indicated the CNTs in composites matrix retained the electronic and structural integrities of the pristine CNTs. Fourier transform infrared (FT-IR), ultraviolet–visible (UV–vis) and circular dichroism (CD) spectroscopy displayed the encapsulated Hb preserved their near-native structure, indicating the polyphosphonate–chitosan–CNTs composites possessed excellent biocompatibility for the encapsulation of proteins/enzymes. Electrochemical measurements indicated the encapsulated Hb could directly exchange electron with the substrate electrode. Moreover, the modified electrode showed excellent bioelectrocatalytic activity for the reduction of hydrogen peroxide. Under optimum experimental conditions, the fabricated electrochemical sensor displayed the fast response (less than 3 s), wide linear range (7.0 × 10⁻⁷ to 2.0 × 10⁻³ M) and low detection limit (4.0 × 10⁻⁷ M) for the determination of hydrogen peroxide. This newly developed protocol was simple and mild and would certainly find extensive applications in biocatalysis, biosensors, bioelectronics and biofuel cells.     

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo⁻), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.     

基于生物条形码的粘蛋白和特定细胞电化学发光适配体生物传感方法研究

本文基于适配体识别和生物条形码放大策略,以MCF-7细胞和粘蛋白(MUC1)为目标物,MUC1的特异性适配体(rcDNA)为分子识别物质,Ru(phen)32+为信号物质,rcDNA通过巯基自组装于金电极表面作为传感界面,发卡DNA (hpDNA)和rcDNA通过巯基自组装在金纳米粒子(AuNP)表面合成的hpDNA/...     

Ultrasensitive detection of DNA by the PCR-Induced generation of DNAzymes: the DNAzyme primer approach

The ultrasensitive detection of DNA is achieved by PCR-induced evolution of a DNAzyme.     

Potentiometric biosensing of proteins with ultrasensitive ion-selective microelectrodes and nanoparticle labels

We report here for the first time on the use of potentiometry for ultrasensitive nanoparticle-based detection of protein interactions. A silver ion-selective microelectrode is used to detect silver ions oxidatively released from silver enlarged gold nanoparticle labels in a sandwich immunoassay. Since potentiometry is expected to largely maintain its analytical characteristics upon reducing the sample volume, it is anticipated that this approach may form the basis for bioassays with attractive detection limits.     

Sensitive and selective DNA detection based on the combination of hairpin-type probe with endonuclease/GNP signal amplification using quartz-crystal-microbalance transduction

In order to develop a highly sensitive and selective piezoelectric transducer for the detection of DNA, the bio-recognizing probe is for the first time designed by introducing a hairpin structure and a recognition site for EcoRI into an oligonucleotide sequence and signal amplifiers are prepared by modifying gold nanoparticles (GNPs) with biomolecules, deepening the application and understanding of biomaterials. The piezoelectric transducer is prepared by immobilizing designed hairpin recognition probe onto the quartz-crystal-microbalance (QCM). In the absence of target DNA, the hairpin probe is removed from the QCM surface after exposure to endonuclease, inhibiting the subsequent signaling reaction. In contrast, introduction of target DNA can open the hairpin probe due to the probe/target hybridization, dissociating the cleavable double-stranded portion. In this case, even if being treated with endonuclease, the integrated hairpin probe is maintained. Subsequent introduction of GNPs modified with detection probes that can hybridize to the terminal sequence of hairpin probe results in a many-folds increase of the frequency response. Utilizing the proposed transduction scheme, the reliable target DNA detection can be accomplished. The detection limit of 2 pM and dynamic response range for target DNA from 2 to 300 pM are obtained. Furthermore, single-base mismatched DNAs can be easily identified. The developed proof-of-principle of a novel piezoelectric transduction scheme is expected to establish a potential platform for the disease-associated mutation analysis and DNA hybridization detection in biotechnology and medical diagnostics.     

Ultrasensitive electrochemical detection of nucleic acid based on the isothermal strand-displacement polymerase reaction and enzyme dual amplification

We describe an ultrasensitive electrochemical detection of DNA protocol based on the isothermal strand-displacement polymerase reaction (ISDPR) and enzyme dual amplifications. Target DNA triggered an ISDPR to produce numerous bi-functionalized duplex DNA complexes. Following an immuno-magnetic collection via an immunoreaction between the attached digoxin on the duplex DNA and the anti-digoxin antibody on the magnetic bead, horseradish (HRP) tracers were bound to the duplex DNA through a biotin–streptavidin interaction. The quantification of DNA was realized by square wave voltammetric detection of the enzymatic products with a screen-printed gold electrode. The voltammetric response was proportional to the concentration of DNA in the range of 0.1 fM–0.5 pM, and the limit of detection was estimated to be 0.06 fM. The new protocol showed great promise for simple, cost-effective, and quantitative gene analysis.