An Autonomous Bio‐barcode DNA Machine for Exponential DNA Amplification and Its Application to the Electrochemical Determination of Adenosine Triphosphate

A novel autonomous bio‐barcode DNA machine that is driven by template‐dependent DNA replication is developed to exponentially amplify special DNA sequences. Combined with a DNA aptamer recognition element, the DNA machine can be further applied in the aptamer‐based, amplified analysis of small molecules. As a model analyte, adenosine triphosphate (ATP) is determined by using the DNA machine system in combination with a DNA aptamer recognition strategy and differential pulse anodic stripping voltammetry (DPASV). Under the optimum conditions, detection limits as low as 2.8×10^?17 M (3σ) for target DNA and 4.7×10^?9 M (3σ) for ATP are achieved. The satisfactory determination of ATP in K562 leukemia cell and Ramos Burkitt’s lymphoma cell reveal that this protocol possesses good selectivity and practicality. As a promising biomolecular device, this DNA machine may have an even broader application in the rapidly developing field of nanobiotechnology.     

Selective Aptamer‐Based Control of Intraneuronal Signaling

Cellular behavior is orchestrated by the complex interactions of a myriad of intracellular signal transduction pathways. To understand and investigate the role of individual components in such signaling networks, the availability of specific inhibitors is of paramount importance. We report the generation and validation of a novel variant of an RNA aptamer that selectively inhibits the mitogen‐activated kinase pathway in neurons. We demonstrate that the aptamer retains function under intracellular conditions and that application of the aptamer through the patch‐clamp pipette efficiently inhibits mitogen‐activated kinase‐dependent synaptic plasticity. This approach introduces synthetic aptamers as generic tools, readily applicable to inhibit different components of intraneuronal signaling networks with utmost specificity.     

Aptamer‐based Biosensor Developed to Monitor MUC1 Released by Prostate Cancer Cells

Electrochemical aptasensors can detect cancer biomarkers such as mucin 1 (MUC1) to provide point‐of‐care diagnosis that is low‐cost, specific and sensitive. Herein, a DNA hairpin containing MUC1 aptamer was thiolated, conjugated with methylene blue (MB) redox tag, and immobilized on a gold electrode by self‐assembly. The fabrication process was characterized by scanning electron microscopy, X‐ray spectroscopy analysis and electrochemistry techniques. The results evidenced a stable and sensitivity sensor presenting wide linear detection range (0.65–110 ng/mL). Therefore, it was able to precisely detect MUC1 production patterns in normal (RWPE‐1) and prostate cancer cells (LNCaP and PC3). The biosensor has ability to detect MUC1 in complex samples being an efficient and useful platform for cancer diagnosing in early stages and for physiological applications such as cancer treatment monitoring.      

Rational Design of a Redox‐Labeled Chiral Target for an Enantioselective Aptamer‐Based Electrochemical Binding Assay

A series of redox‐labeled L ‐tyrosinamide (L ‐Tym) derivatives was prepared and the nature of the functional group and the chain length of the spacer were systematically varied in a step‐by‐step affinity optimization process of the tracer for the L ‐Tym aptamer. The choice of the labeling position on L ‐Tym proved to be crucial for the molecular recognition event, which could be monitored by cyclic voltammetry and is based on the different diffusion rates of free and bound targets in solution. From this screening approach an efficient electroactive tracer emerged. Comparable dissociation constants K_d were obtained for the unlabeled and labeled targets in direct or competitive binding assays. The enantiomeric tracer was prepared and its enantioselective recognition by the corresponding anti‐D ‐Tym aptamer was demonstrated. The access to both enantiomeric tracer molecules opens the door for the development of one‐pot determination of the enantiomeric excess when using different labels with well‐separated redox potentials for each enantiomer.     

[Ru(bpy)2(dcbpy)NHS] Labeling/Aptamer‐Based Biosensor for the Detection of Lysozyme by Increasing Sensitivity with Gold Nanoparticle Amplification

A novel [Ru(bpy)₂(dcbpy)NHS] labeling/aptamer‐based biosensor combined with gold nanoparticle amplification for the determination of lysozyme with an electrochemiluminescence (ECL) method is presented. In this work, an aptamer, an ECL probe, gold nanoparticle amplification, and competition assay are the main protocols employed in ECL detection. With all the protocols used, an original biosensor coupled with an aptamer and [Ru(bpy)₂(dcbpy)NHS] has been prepared. Its high selectivity and sensitivity are the main advantages over other traditional [Ru(bpy)₃]²⁺ biosensors. The electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) characterization illustrate that this biosensor is fabricated successfully. Finally, the biosensor was applied to a displacement assay in different concentrations of lysozyme solution, and an ultrasensitive ECL signal was obtained. The ECL intensity decreased proportionally to the lysozyme concentration over the range 1.0×10^?13–1.0×10^?8 mol L^?1 with a detection limit of 1.0×10^?13 mol L^?1. This strategy for the aptasensor opens a rapid, selective, and sensitive route for the detection of lysozyme and potentially other proteins.     

Aptamer‐Based SERS Assay of ATP and Lysozyme by Using Primer Self‐Generation

A simple bifunctional surface‐enhanced Raman scattering (SERS) assay based on primer self‐generation strand‐displacement polymerization (PS‐SDP) is developed to detect small molecules or proteins in parallel. Triphosphate (ATP) and lysozyme are used as the models of small molecules and proteins. Compared to traditional bifunctional methods, the method possesses some remarkable features as follows: 1) by virtue of the simple PS‐SDP reaction, a bifunctional aptamer assembly binding of trigger 1 and trigger 2 was used as a functional structure for the simultaneous sensing of ATP or lysozyme. 2) The concept of isothermal amplification bifunctional detection has been first introduced into SERS biosensing applications as a signal‐amplification tool. 3) The problem of high background induced by excess bio‐barcodes is circumvented by using magnetic beads (MBs) as the carrier of signal‐output products and massive of hairpin DNA binding with SERS active bio‐barcodes relied on Au nanoparticles (Au NPs), SERS signal is significantly enhanced. Overall, with multiple amplification steps and one magnetic‐separation procedure, this flexible biosensing system exhibited not only high sensitivity and specificity, with the detection limits of ATP and lysozyme of 0.05 nM and 10 fM , respectively.     

A Universal Strategy for Aptamer‐Based Nanopore Sensing through Host–Guest Interactions inside α‐Hemolysin

Nanopore emerged as a powerful single‐molecule technique over the past two decades, and has shown applications in the stochastic sensing and biophysical studies of individual molecules. Here, we report a versatile strategy for nanopore sensing by employing the combination of aptamers and host–guest interactions. An aptamer is first hybridized with a DNA probe which is modified with a ferrocene?cucurbit[7]uril complex. The presence of analytes causes the aptamer–probe duplex to unwind and release the DNA probe which can quantitatively produce signature current events when translocated through an α‐hemolysin nanopore. The integrated use of magnetic beads can further lower the detection limit by approximately two to three orders of magnitude. Because aptamers have shown robust binding affinities with a wide variety of target molecules, our proposed strategy should be universally applicable for sensing different types of analytes with nanopore sensors.     

Bio‐Inspired Fabrication of Lotus Leaf Like Membranes as Fluorescent Sensing Materials

A fluorescent organic small molecule, hexaphenylsilole (HPS), has been used as a sensing material, while a HPS/polymethyl methacrylate composite film with a lotus leaf like structure is prepared by a simple electrospin method. The film shows high stability and excellent sensitivity for the metal ions Fe³⁺ and Hg²⁺, respectively. The special surface morphology containing micro‐/nanocomposite structure is attributed to the exhibition of these unusual properties.     

ADLOC: An Aptamer‐Displacement Assay Based on Luminescent Oxygen Channeling

Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high‐throughput screening. Here we describe an unprecedented type of a nucleic acid‐based sensor system and show that it is amenable to high‐throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof‐of‐principle study we demonstrate that the format is feasible for efficient identification of small drug‐like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin‐specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA‐aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer‐displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18 000 drug‐like small molecules two compounds as strong singlet‐oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC‐based assay described here could be used to screen at least 100 000 compounds per day.     

A Rapid and Sensitive Aptamer‐Based Electrochemical Biosensor for Direct Detection of Escherichia Coli O111

A sensitive and specific electrochemical biosensor based on target‐induced aptamer displacement was developed for direct detection of Escherichia coli O111. The aptamer for Escherichia coli O111 was immobilized on a gold electrode by hybridization with the capture probe anchored on the electrode surface through Au‐thiol binding. In the presence of Escherichia coli O111, the aptamer was dissociated from the capture probe‐aptamer duplex due to the stronger interaction between the aptamer and the Escherichia coli O111. The consequent single‐strand capture probe could be hybridized with biotinylated detection probe and tagged with streptavidin‐alkaline phosphatase, producing sensitive enzyme‐catalyzed electrochemical response to Escherichia coli O111. The designed biosensor showed weak electrochemical signal to Salmonella typhimurium, Staphylococcus aureus and common non‐pathogenic Escherichia coli, indicating high specificity for Escherichia coli O111. Under the optimal conditions, the proposed strategy could directly detect Escherichia coli O111 with the detection limit of 112 CFU mL^?1 in phosphate buffer saline and 305 CFU mL^?1 in milk within 3.5 h, demonstrated the sensitive and accurate quantification of target pathogenic bacteria. The designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.     

Recent Progress in Aptamer‐Based Functional Probes for Bioanalysis and Biomedicine

Nucleic acid aptamers are short synthetic DNA or RNA sequences that can bind to a wide range of targets with high affinity and specificity. In recent years, aptamers have attracted increasing research interest due to their unique features of high binding affinity and specificity, small size, excellent chemical stability, easy chemical synthesis, facile modification, and minimal immunogenicity. These properties make aptamers ideal recognition ligands for bioanalysis, disease diagnosis, and cancer therapy. This review highlights the recent progress in aptamer selection and the latest applications of aptamer‐based functional probes in the fields of bioanalysis and biomedicine.     

3,4,9,10‐Perylenetetracarboxylic Acid/Hemin Nanocomposites Act as Redox Probes and Electrocatalysts for Constructing a Pseudobienzyme‐Channeling Amplified Electrochemical Aptasensor

A simple wet‐chemical strategy for the synthesis of 3,4,9,10‐perylenetetracarboxylic acid (PTCA)/hemin nanocomposites through π–π interactions is demonstrated. Significantly, the hemin successfully conciliates PTCA redox activity with a pair of well‐defined redox peaks and intrinsic peroxidase‐like activity, which provides potential application of the PTCA self‐derived redox activity as redox probes. Additionally, PTCA/hemin nanocomposites exhibit a good membrane‐forming property, which not only avoids the conventional fussy process for redox probe immobilization, but also reduces the participation of the membrane materials that act as a barrier of electron transfer. On the basis of these unique properties, a pseudobienzyme‐channeling amplified electrochemical aptasensor is developed that is coupled with glucose oxidase (GOx) for thrombin detection by using PTCA/hemin nanocomposites as redox probes and electrocatalysts. With the addition of glucose to the electrolytic cell, the GOx on the aptasensor surface bioelectrocatalyzed the reduction of glucose to produce H₂O₂, which in turn was electrocatalyzed by the PTCA/hemin nanocomposites. Cascade schemes, in which an enzyme is catalytically linked to another enzyme, can produce signal amplification and therefore increase the biosensor sensitivity. As a result, a linear relationship for thrombin from 0.005 to 20 nM and a detection limit of 0.001 nM were obtained.     

Self‐Assembled Aptamer‐Based Drug Carriers for Bispecific Cytotoxicity to Cancer Cells

Monovalent aptamers can deliver drugs to target cells by specific recognition. However, different cancer subtypes are distinguished by heterogeneous biomarkers and one single aptamer is unable to recognize all clinical samples from different patients with even the same type of cancers. To address heterogeneity among cancer subtypes for targeted drug delivery, as a model, we developed a drug carrier with a broader recognition range of cancer subtypes. This carrier, sgc8c‐sgd5a (SD), was self‐assembled from two modified monovalent aptamers. It showed bispecific recognition abilities to target cells in cell mixtures; thus broadening the recognition capabilities of its parent aptamers. The self‐assembly of SD simultaneously formed multiple drug loading sites for the anticancer drug doxorubicin (Dox). The Dox‐loaded SD (SD–Dox) also showed bispecific abilities for target cell binding and drug delivery. Most importantly, SD–Dox induced bispecific cytotoxicity in target cells in cell mixtures. Therefore, by broadening the otherwise limited recognition capabilities of monovalent aptamers, bispecific aptamer‐based drug carriers would facilitate aptamer applications for clinically heterogeneous cancer subtypes that respond to the same cancer therapy.     

Synthesis of Bismuth‐Nanoparticle‐Enriched Nanoporous Carbon on Graphene for Efficient Electrochemical Analysis of Heavy‐Metal Ions

A BiNPs@NPCGS nanocomposite was designed for highly efficient detection of multiple heavy‐metal ions by in situ synthesis of bismuth‐nanoparticle (BiNP)‐enriched nanoporous carbon (NPS) on graphene sheet (GS). The NPCGS was prepared by pyrolysis of zeolitic imidazolate framework‐8 (ZIF‐8) nanocrystals deposited on graphene oxide and displayed a high surface area of 1251 m² g^?1 and a pore size of 3.4 nm. BiNPs were deposited on NPCGS in situ by chemical reduction of Bi³⁺ with NaBH₄. Due to the restrictive effect of the pore/surface structure of NPCGS, the BiNPs were uniform and well dispersed on the NPCGS. The BiNPs@NPCGS showed good conductivity and high effective area, and the presence of BiNPs allowed it to act as an efficient material for anodic‐stripping voltammetric detection of heavy‐metal ions. Under optimized conditions, the BiNPs@NPCGS‐based sensor could simultaneously determine Pb²⁺ and Cd²⁺ with detection limits of 3.2 and 4.1 nM , respectively. Moreover, the proposed sensor could also differentiate Tl⁺ from Pb²⁺ and Cd²⁺. Owing to its advantages of simple preparation, environmental friendliness, high surface area, and fast electron‐transfer ability, BiNPs@NPCGS showed promise for practical application in sensing heavy‐metal ions.     

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.