Resonance scattering spectral detection of trace ATP based on label-free aptamer reaction and nanogold catalysis

Double-stranded DNA (dsDNA) cannot protect gold nanoparticles (AuNPs) in the presence of NaCl, and dsDNA interacted with adenosine triphosphate (ATP) to form stable G-quartet and a single-stranded DNA (DNA 2) that can protect AuNPs. The unprotected AuNPs were aggregated to AuNP aggregations (AuNPA) that exhibited a resonance scattering (RS) peak at 590 nm. The RS intensity at 590 nm decreased linearly when the ATP concentration increased in the range of 6.6-110 nM. The catalysis of AuNP-DNA 2 was stronger than that of the AuNPA on the glucose-Cu(II) particle reaction, and the product appeared as an RS peak at 620 nm. When the ATP concentration was increased, the AuNP-DNA 2 increased, and the RS intensity at 620 nm increased linearly. The increased RS intensity (ΔI(620 nm)) was linear to ATP concentration in the range of 2.2-220 nM, with a regression equation of ΔI(620 nm) = 0.709C + 7.7, and a detection limit of 0.5 nM. Hereby, a new RS method of ATP detection was set up with high sensitivity and selectivity.     

A new method for the detection of ATP using a quantum-dot-tagged aptamer

Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection. Zhang Chen and Guang Li contributed equally to this work.     

A label-free electrochemical biosensor based on a DNA aptamer against codeine

In order to develop a sensor for opium alkaloid codeine detection, DNA aptamers against codeine were generated by SELEX (systematic evolution of ligands by exponential enrichment) technique. An aptamer HL7-14, which is a 37-mer sequence with K_d values of 0.91 μM, was optimized by the truncation-mutation assay. The specificity investigation shows that HL7-14 exhibits high specificity to codeine over morphine, and almost cannot bind to other small molecule. With this new selected aptamer, a novel electrochemical label-free codeine aptamer biosensor based on Au-mesoporous silica nanoparticles (Au-MSN) as immobilized substrate has been proposed using [Fe(CN)₆]^3−/4− as electroactive redox probe. The linear range covered from 10 pM to 100 nM with correlation coefficient of 0.9979 and the detection limit was 3 pM. Our study demonstrates that the biosensor has good specificity, stability and well regeneration. It can be used to detect codeine.     

A label-free electrochemical RNA aptamer for selective detection of theophylline

In this work, we report a novel electrochemical RNA aptamer for the selective detection of theophylline. Firstly, gold nanoparticles were electrodeposited on the surface of glassy carbon (GC) electrode to form a gold nanoparticles modified electrode. Secondly, the designed single-stranded RNA (ssRNA) was immobilized on gold nanoparticles through a thiol linker as a probe RNA. Then, the complement stranded RNA, which can combine with the probe ssRNA to form a double-stranded RNA (dsRNA) with a recognition unit of theophylline, was linked on the probe RNA through a hybrid reaction in the presence of theophylline. Doxorubicin was selected as an electrochemical indicator. The proposed RNA aptamer presents an excellent selectivity for the detection of theophylline. The detectable concentration range of theophylline is from 2.0 to 50.0 μM with a limit of detection of 1.2 μM.     

Amplified label-free electrical detection of DNA hybridization

A new protocol is described for amplifying label-free electrochemical measurements of DNA hybridization based on the enhanced accumulation of purine nucleobases in the presence of copper ions . Such electrical DNA assays involve hybridization of the target to inosine-substituted oligonucleotide probes (captured on magnetic beads), acidic dipurinization of the hybrid DNA, and adsorptive chronopotentiometric stripping measurements of the free nucleobases in the presence of copper ions. Both amplified adenine and guanine peaks can be used for detecting the DNA hybridization. The dramatic signal amplification advantage of this type of detection has been combined with efficient magnetic removal of non-complementary DNA, use of microliter sample volumes and disposable transducers. Factors influencing the signal enhancement were assessed and optimized. A detection limit of 40 fmol (250 pg) was obtained with 10 min hybridization and 5 min adsorptive-accumulation times. The advantages of this procedure were demonstrated by its application in the detection of DNA segments related to the BRCA1 breast cancer gene. The copper enhancement holds great promise not only for the detection of DNA hybridization, but also for trace measurement of nucleic acids.     

Turbidimetric detection of ATP using polymeric micelles and DNA aptamers

Two types of turbidimetric detection of adenosine 5'-triphosphate (ATP) by the naked eye were achieved through a combination of non-cross-linking aggregation of DNA-linked polymeric micelles and molecular recognition of ATP by a DNA aptamer.     

A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN)₆]^3−/4−. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.     

Highly sensitive label-free in vitro detection of aflatoxin B1 in an aptamer assay using optical planar waveguide operating as a polarization interferometer

Analytical and Bioanalytical Chemistry - This work reports on further development of an optical biosensor for the in vitro detection of mycotoxins (in particular, aflatoxin B1) using a highly...     

Nanomolar fluorescent detection of c-di-GMP using a modular aptamer strategy

C-di-GMP regulates important processes involved in biofilm formation and virulence factors production in several bacteria. Herein we report a simple fluorescent strategy that allows for the detection of c-di-GMP (as low as 320 nM) using a Vc2 class I riboswitch domain as the sensing region and spinach as the fluorescent reporting module.     

A label-free G-quadruplex-based switch-on fluorescence assay for the selective detection of ATP

A G-quadruplex-based, label-free, switch-on fluorescence detection method has been developed for the selective detection of ATP in aqueous solution using crystal violet as a G-quadruplex-selective probe. The assay is highly simple and rapid, and does not require the use of fluorescent labeling.     

A label-free fluorescent turn-on enzymatic amplification assay for DNA detection using ligand-responsive G-quadruplex formation

A facile and general label-free assay for sensitive and selective DNA detection has been developed based on enzyme amplification and ligand-responsive quadruplex formation.     

Magnetic bead-based label-free electrochemical detection of DNA hybridization.

Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.     

Enhanced catalytic DNAzyme for label-free colorimetric detection of DNA

A DNAzyme-based label-free method for the colorimetric detection of DNA is introduced, with a supramolecular hemin-G-quartet complex as the sensing element and a 36-mer single-strand DNA as the analyte that is detected at 10 nM.     

Quantitative label-free RNA detection using surface-enhanced Raman spectroscopy

Surface-Enhanced Raman Spectroscopy (SERS) was performed to detect label-free RNA. We defined conditions which make it possible to probe the four bases of RNA, in single strands of polyadenosine (pA), polyuridine (pU), polycytosine (pC) and polyguanosine (pG). We therefore present below a quantitative analysis of mixtures of non-hybridized single strands, based on the deconvolution of the SERS mixture spectrum into the relative contributions of the SERS spectra of each constituent.     

Exonuclease-assisted cascaded recycling amplification for label-free detection of DNA

The network consisting of three kinds of unlabeled stem-loop DNA molecular beacons (MBs) is activated by target DNA in the presence of exonuclease-III (Exo-III), achieving the concept of exonuclease-assisted cascaded recycling amplification (Exo-CRA) for DNA detection with a wide dynamic range of 8 orders of magnitude.     

Rapid detection of a cocaine-binding aptamer using biological nanopores on a chip

This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.     

基于核酸适体的丙肝病毒核心蛋白检测新方法

丙型病毒性肝炎是由丙型肝炎病毒(hepatitis C virus,HCV)引起的一种传染性疾病.发展对HCV抗原具有高亲和力高特异性的识别分子和检测方法对丙肝进行早期诊断具有非常重要的意义.我们通过SELEX筛选得到了能特异性识别HCV核心蛋白(core蛋白)的DNA核酸适体,建立了可对HCV core蛋白进行高灵敏检测的核酸适体-酶联免疫新方法,并成功应用于丙肝病人血清中HCV core蛋白的检测,有望发展成为简便、灵敏、低成本的HCV早期诊断和血清筛查新方法.     

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-G_NP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G_NP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G_NP were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)₆]^3−/4− as the redox indicator. The (CA-G_NP/SPAN)_n films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-G_NP) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)₆]^3−/4− in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-G_NP/SPAN)_n films, the target DNA with 20-base could be detected up to 2.13 × 10⁻¹³ mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated.