A novel label-free electrochemical aptasensor for thrombin based on the {nano-Au/thionine}n multilayer films as redox probes

Herein, a novel label-free electrochemical aptasensor based on direct immobilization of the redox probes on an electrode surface was reported. Gold electrode coated Nafion was firstly modified with redox probe-thionine (Thi) through ion exchange adsorption. Then, with the help of chemisorption and electrostatic adsorption, negatively charged nano-Au and positively charged Thi were layer-by-layer (LBL) self-assembled onto the modified electrode surface, which formed {nano-Au/Thi⁺}_n multilayer films for improving the amount of redox probes and immobilizing thiolated thrombin aptamers (TBA). In the presence of target thrombin (TB), the TBA on the multilayer film could catch the TB onto the electrode surface, which resulted in a barrier for electro-transfer, leading to decrease of the current. The proposed method avoided the cubsome redox probe labeling process, increased the amount of redox probe and reduced the distance between the redox probe and electrode surface. Thus, the approach showed a high sensitivity and a wider linearity to TB in the range from 0.12 nM to 46 nM with a detection limit of 40 pM.     

Heterogeneous and Homogeneous Aptamer‐Based Electrochemical Sensors for Thrombin

Nanomolar concentrations of thrombin were electrochemically monitored using heterogeneous switch‐on and homogeneous switch‐off approaches that incorporated ferrocenyl aptamers. For the first time, the heterogeneous approach was coupled to a glucose/glucose oxidase (GOx) amplification‐regeneration system which increased its sensitivity by 2 folds with detection limits of 4.3 nM and 2.5 nM in the absence and presence of glucose/GOx, respectively. We also present a new homogeneous system involving the ferrocenyl aptamer binding thrombin in solution causing a significant decrease in its diffusion coefficient. Thus the ferrocene anodic current decreased at an unmodified gold electrode with detection limit of 3.9 nM and 12 times larger linear range than the heterogeneous method.     

Highly sensitive determination of recombinant human erythropoietin-α in aptamer-based affinity probe capillary electrophoresis with laser-induced fluorescence detection

Recombinant human erythropoietin-α (rHuEPO-α) has been widely used in clinic for anemia treatment. The detection and quantification of rHuEPO-α is essential for monitoring this widespread recombinant glycoprotein pharmaceutical. In this paper, we developed a new affinity probe capillary electrophoresis/laser-induced fluorescence (APCE/LIF) method for the detection of rHuEPO-α by using a specific single-stranded DNA aptamer probe for the first time. In this method, the complex of aptamer-rHuEPO-α and the free aptamer can be well separated and identified by their migration and fluorescence intensity after systematic optimization. The existence of sodium cation in the sample buffer and running buffer played a critical role for stabilizing complex and enhancing the separation efficiency, additionally, suitable high voltage and sample buffer additives were also important for improving the peak height of the complex. Under the optimized conditions, the method was successfully applied for the quantification of rHuEPO-α in physiological buffer, artificial urine and human serum. The linear range for rHuEPO-α was from 0.2 to 100 nM and the limit of detection was 0.2 nM (i.e. 7.4 ng/mL). Further binding experiments using fluorescein isothiocyanate-labeled rHuEPO-α (F-rHuEPO-α) and N-deglycosylated F-rHuEPO-α demonstrated that the oligosaccharides moiety was of importance in the specific interaction between rHuEPO-α and its aptamer.     

Aptamer-oligonucleotide binding studied by capillary electrophoresis: cation effect and separation efficiency

Novel features of DNA structure, recognition and discrimination have been recently elucidated through the solution structural characterization of DNA aptamers that bind cofactors, amino acids and peptides with high affinity and specificity. Multidimensional nuclear magnetic resonance methodologies have been successfully applied to solve the solution structures. In this work, it was demonstrated that capillary electrophoresis was a powerful tool allowing the fundamental study of the binding mechanism between a DNA aptamer and three ligands, adenosine and adenylate compounds, i.e., adenosine diphosphate (ADP) and adenosine triphosphate (ATP). In order to gain further insight into this binding, thermodynamic measurements under different values of parameters (such as salt nature and its concentration (x) in the run buffer) were carried out. The results showed that dehydration at the binding interface, van der Waals interactions, H-bonding and adjustment of the aptamer recognition surface were implied in the aptamer-ligand association. As well, it was demonstrated that the addition in the medium of the sodium monovalent cation Na(+) or the nickel divalent cation Ni(2+) decreased the complex formation. Separation efficiency and peak shape can also be improved by Mg(2+) divalent cation, which increased the mass transfer kinetics during the ligand-aptamer binding process. A significant separation for the worst separated pair of peaks on the electropherogram ((ADP, ATP) peak pair) was thus achieved.     

Aptamer-Based Alternatives to the Conventional Immobilized Metal Affinity Chromatography for Purification of His-Tagged Proteins

Aptamers are oligonucleotides or peptide molecules that are able to bind to their specific target molecules with high affinity via molecular recognition. In this study, we present development of aptamer-based affinity purification for His-tagged proteins for comparison of purification efficiency with the conventional Ni²⁺-based affinity chromatography. Thiol-functionalized aptamers able to specifically bind to His-tag were immobilized employing two crosslinking methods onto the surface of polystyrene resins. The resulting aptamer-anchored resins were successfully applied for purification of His-tagged proteins from complex E. coli and human cell lysates, respectively, and superior or at least comparable purification results to the conventional immobilized metal affinity chromatography were obtained via one-step purification.     

A novel ratiometric fluorescent aptasensor accurately detects patulin contamination in fruits and fruits products

Patulin (PAT) contamination in fruit and fruit products is a significant public health concern. Here, we developed a ratiometric fluorescent aptasensor for PAT detection based on aptamer-recognition and Exonuclease III amplification. Two structure selective dyes, SYBR Green I (SGI) and N-methyl mesoporphyrin IX (NMM), were used as fluorescent probes. In the developed biosensing system, the binding of PAT to aptamer triggered the liberation of cDNA. Subsequently, amplification was mediated by Exonuclease III. S1 was released from the S1-S2 duplex by enzymatic hydrolyzation and incorporated into a stable G-quadruplex. As a result, the fluorescence of SGI decreased, whereas that of NMM increased. There was a strong linear correlation between the relative fluorescence intensity and PAT concentrations (20 to 500 ng·L^?1 range) (R² = 0.99). The biosensing system was highly sensitive, and could detect PAT concentration as low as 4.7 ng·L^?1. The sensor was also highly specific, and could differentiate PAT from several other related mycotoxins. In summary, we developed a new bioassay for the accurate detection of PAT contamination in fruits and fruit products. This research provides a new approach for developing ratiometric bioassays based on structure-selective dyes and enzymatic conversion processes.     

Aptamer enzymatic cleavage protection assay for the gold nanoparticle-based colorimetric sensing of small molecules

A label-free, homogeneous aptamer-based sensor strategy was designed for the facile colorimetric detection of small target molecules. The format relied on the target-induced protection of DNA aptamer from the enzymatic digestion and its transduction into a detectable signal through the length-dependent adsorption of single-stranded DNA onto unmodified gold nanoparticles (AuNPs). The proof-of-principle of the approach was established by employing the anti-tyrosinamide aptamer as a model functional nucleic acid. In the absence of target, the aptamer was cleaved by the phosphodiesterase I enzymatic probe, leading to the release of mononucleotides and short DNA fragments. These governed effective electrostatic stabilization of AuNPs so that the nanoparticles remained dispersed and red-colored upon salt addition. Upon tyrosinamide binding, the enzymatic cleavage was impeded, resulting in the protection of the aptamer structure. As this long DNA molecule was unable to electrostatically stabilize AuNPs, the resulting colloidal solution turned blue after salt addition due to the formation of nanoparticle aggregates. The quantitative determination of the target can be achieved by monitoring the ratio of absorbance at 650 and 520 nm of the gold colloidal solution. A limit of detection of ∼5 μM and a linear range up to 100 μM were obtained. The sensing platform was further applied, through the same experimental protocol, to the adenosine detection by using its DNA aptamer as recognition tool. This strategy could extend the potentialities, in terms of both simplicity and general applicability, of the aptamer-based sensing approaches.     

Fast functionalization of silver decahedral nanoparticles with aptamers for colorimetric detection of human platelet-derived growth factor-BB

Aptamer-silver decahedral nanoparticles (Ag₁₀NPs-aptamer) based detection was developed for protein. Ag₁₀NPs were synthesized by photochemical method. The advantage of Ag₁₀NPs was its tolerance of NaCl which facilitates the functionalization of silver nanoparticles with all kinds of ssDNA. Attaching aptamers to Ag₁₀NPs could be achieved within 2 h, much faster than traditional methods. Human platelet-derived growth factor-BB (PDGF-BB) was used as a model protein to test the binding capacity of aptamers attached on Ag₁₀NPs. Our data showed that the aptamer-Ag₁₀NPs conjugates were successful in detecting human PDGF-BB. Furthermore, we developed an aptamer-Ag₁₀NPs conjugates-based colorimetric sensor to detect PDGF-BB. The results showed a linear relationship between PDGF-BB concentrations (5 ng mL⁻¹–200 ng mL⁻¹) and ΔOD with excellent detection specificity in serum. Therefore, the sensor based on aptamer-Ag₁₀NPs conjugates was highly effective and sensitive and had great promise for further development and applications.     

Aptamer-based homogeneous protein detection using cucurbit[7]uril functionalized electrode

A new strategy for homogeneous protein detection is developed based on a cucurbit[7]uril (CB[7]) functionalized electrode. The analytical procedure consists of the binding of target protein to its aptamer in the test solution, followed by an exonuclease-catalyzed digestion of methylene blue (MB) tag labeled DNA oligonucleotides. Since CB[7] molecules immobilized on the electrode may efficiently capture the released MB-labeled nucleotides, the MB tags are concentrated to the electrode surface and subsequently yield highly sensitive electrochemical signal, which is related to the concentration of the target protein. The method combines the host–guest properties of CB[7] with the immobilization-free homogeneous assay, providing a powerful tool for protein detection. Taking the detection of osteopontin as an example, the proposed method can have a linear response to the target protein in a range from 50 to 500 ng mL⁻¹ with a detection limit of 10.7 ng mL⁻¹. It can also show high specificity and good reproducibility, and can be used directly for the assay of osteopontin in serum samples.     

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.