Optical and Electrochemical Probes for Monitoring Cytochrome c in Subcellular Compartments During Apoptosis

Cytochrome c (Cyt. c) is a key initiator of the caspases that activate cell apoptosis. The spatiotemporal evaluation of the contents of Cyt. c in cellular compartments and the detection of Cyt. c delivery between cellular compartments upon apoptosis is important for probing cell viabilities. We introduce an optical probe and an electrochemical probe for the quantitative assessment of Cyt. c in cellular compartments at the single cell level. The optical or electrochemical probes are functionalized with photoresponsive o-nitrobenzylphosphate ester-caged Cyt. c aptamer constituents. These are uncaged by light stimuli at single cell compartments, allowing the spatiotemporal detection of Cyt. c through the formation of Cyt. c/aptamer complexes at non-apoptotic or apoptotic conditions. The probes are applied to distinguish the contents of Cyt. c in cellular compartments of epithelial MCF-10A breast cells and malignant MCF-7 and MDA-MB-231 breast cells under apoptotic/non-apoptotic conditions.     

Competitive Assay for Theophylline Based on an Abasic Site‐Containing DNA Duplex Aptamer and a Fluorescent Ligand

A fluorescence assay for theophylline, one of the common drugs for acute and chronic asthmatic conditions, has been developed based on an abasic site‐containing DNA duplex aptamer (AP aptamer) in combination with an abasic site‐binding fluorescent ligand, riboflavin. The assay is based on the competitive binding of theophylline and riboflavin at the abasic (AP) site of the AP aptamer. In the absence of theophylline, riboflavin binds to the receptor nucleotide opposite the AP site, which leads to fluorescence quenching of the riboflavin. Upon addition of theophylline, competitive binding occurs between theophylline and riboflavin, which results in an effective fluorescence restoration due to release of riboflavin from the AP site. From an examination of the optimization of the AP aptamers, the complex of riboflavin with a 23‐mer AP aptamer (5′‐TCT GCG TCC AGX GCA ACG CAC AC‐3′/5′‐GTG TGC GTT GCC CTG GAC GCA GA‐3′; X : the AP site (Spacer C3, a propylene residue)) possessing cytosine as a receptor nucleotide was found to show a selective and effective fluorescence response to theophylline; the limit of detection for theophylline was 1.1 μM . Furthermore, fluorescence detection of theophylline was successfully demonstrated with high selectivity in serum samples by using the optimized AP aptamer and riboflavin.     

Determination of binding parameters between lysozyme and its aptamer by frontal analysis continuous microchip electrophoresis (FACMCE)

An original and simple methodology based on microchip electrophoresis (MCE) in a continuous frontal analysis mode (named frontal analysis continuous microchip electrophoresis, FACMCE) was developed for the simultaneous determination of the binding parameters, i.e. ligand-site dissociation constant (k(d)) and number of binding sites on the substrate (n). This simultaneous determination was exemplified with the interaction between an aptamer and its target. The selected target is a strongly basic protein, lysozyme, as its quantification is of great interest due to its antimicrobial and allergenic properties. A glass microdevice equipped with a fluorescence detection system was coated with hydroxypropylcellulose, reducing the electroosmotic flow and adsorption onto the channel walls. This microdevice allowed the continuous electrokinetic injection of a mixture of fluorescently labelled aptamer and non-labelled lysozyme. By determining the concentration of the free fluorescently labelled aptamer thanks to its corresponding plateau height, mathematical linearization methods allowed to determine a k(d) value of 48.4±8.0 nM, consistent with reported results (31 nM), while the average number of binding sites n on lysozyme, never determined before, was 0.16±0.03. These results seem to indicate that the buffer nature and the SELEX process should influence the number and affinity of the binding sites. In parallel it has been shown that the binding between lysozyme and its aptamer presents two sites of different binding affinities.     

Aptamer-antibody sandwich assay for cytochrome c employing an MWCNT platform and electrochemical impedance

We report on a sensitive aptamer-antibody interaction-based assay for cytochrome c (Cyt c) using electrochemical impedance. 4-Amino benzoic acid is used for the oriented immobilization of aminated aptamers onto multi-walled carbon nanotubes on the surface of a screen-printed electrode via electrochemical grafting. Impedance was measured in a solution containing the redox system ferro/ferricyanide. The change in interfacial charge transfer resistance (R_ct) experienced by the redox marker was recorded to confirm the formation of a complex between aptamer and the target (Cyt c). A biotinylated antibody against cytochrome c was then used in a sandwich type of assay. The addition of streptavidin conjugated to gold nanoparticles and signal enhancement by treatment with silver led to a further increase in Rct. Under optimized conditions, a detection limit as low as 12 pM was obtained. Cross-reactivity against other serum proteins including fibrinogen, BSA and immunoglobulin G demonstrated improved selectivity.

Sensitive and selective assay for cytochrome c protein using aptamer linked to multi-walled carbon nanotube screen printed electrode via diazonium electrochemical grafting and specific biotinylated antibody to improve selectivity. Detection can be based on electrochemical impedance spectroscopy, or using a streptavidin-gold nanoparticle conjugate.

     

Specific Binding of a d‐RNA G‐Quadruplex Structure with an l‐RNA Aptamer

G‐quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4‐binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l ‐RNA aptamer that targets a d ‐RNA G‐quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l ‐RNA aptamer, Ap3‐7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA‐rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4‐specific tools for diverse applications.     

Electrochemiluminescent aptamer biosensor for the determination of ochratoxin A at a gold-nanoparticles-modified gold electrode using N-(aminobutyl)-N-ethylisoluminol as a luminescent label

A highly selective electrochemiluminescent biosensor for the detection of target nephrotoxic toxin, ochratoxin A (OTA), was developed using a DNA aptamer as the recognition element and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) as the signal-producing compound. The electrochemiluminescent aptamer biosensor was fabricated by immobilizing aptamer complementary DNA 1 sequence onto the surface of a gold-nanoparticle (AuNP)-modified gold electrode. ABEI-labeled aptamer DNA 2 sequence hybridized to DNA 1 and was utilized as an electrochemiluminescent probe. A decreased electrochemiluminescence (ECL) signal was generated upon aptamer recognition of the target OTA, which induced the dissociation of DNA 2 (ABEI-labeled aptamer electrochemiluminescent probe) from DNA 1 and moved it far away from the electrode surface. Under the optimal conditions, the decreased ECL intensity was proportional to an OTA concentration ranging from 0.02 to 3.0 ng mL^-1, with a detection limit of 0.007 ng mL^-1. The relative standard deviation was 3.8% at 0.2 ng mL^-1 (n = 7). The proposed method has been applied to measure OTA in naturally contaminated wheat samples and validated by an official method. This work demonstrates the combination of a highly binding aptamer with a highly sensitive ECL technique to design an electrochemiluminescent biosensor, which is a very promising approach for the determination of small-molecule toxins.     

Specific Binding of a d-RNA G-Quadruplex Structure with an l-RNA Aptamer

G-quadruplex (G4) structures are of general importance in chemistry and biology, such as in biosensing, gene regulation, and cancers. Although a large repertoire of G4-binding tools has been developed, no aptamer has been developed to interact with G4. Moreover, the G4 selectivity of current toolkits is very limited. Herein, we report the first l -RNA aptamer that targets a d -RNA G-quadruplex (rG4). Using TERRA rG4 as an example, our results reveal that this l -RNA aptamer, Ap3-7, folds into a unique secondary structure, exhibits high G4 selectivity and effectively interferes with TERRA-rG4–RHAU53 binding. Our approach and findings open a new door in further developing G4-specific tools for diverse applications.     

Direct Electrochemistry of Cytochrome c Based on Poly(diallyldimethylammonium Chloride)‐ Graphene Nanosheets/Gold Nanoparticles Hybrid Nanocomposites and Its Biosensing

A novel biosensor was developed by entrapping cytochrome c (Cyt c) in thin films of the room temperature ionic liquid (RTIL) containing nanocomposites of poly(diallyldimethylammonium chloride)‐graphene nanosheets‐gold nanoparticles (PDDA‐Gp‐AuNPs) at a 11‐mercaptoundecanoic acid‐6‐mercapto‐1‐hexanol modified gold electrode. The synthesized PDDA‐Gp‐AuNPs hybrid nanocomposites were characterized by UV‐vis spectroscopy, Raman spectroscopy, scanning electron microscopy and atomic force microscopy. The PDDA‐Gp‐AuNPs nanocomposites could increase the effective surface of the electrode, enhance the fixed amount of Cyt c on the electrode surface, promote the electron transfer and facilitate the catalytic activity of Cyt c. The RTIL could provide a biocompatible microenvironment to keep Cyt c biological activities, act as an effective mediator to immobilize a large number of Cyt c on the electrode and have good conductivity to improve electron transfer. Therefore, the resultant electrode exhibited good electrochemical performance and electrocatalytic activity. It could be used for electrochemical detection of H₂O₂ with rapid response, high sensitivity, wide linear range and low detection limit, as well as good stability, repeatability and selectivity. The sensor might be promising for practical application.     

Aptamer-based electrochemical biosensor for <Emphasis Type="Italic">Botulinum</Emphasis> neurotoxin

We have developed an aptamer-based electrochemical sensor for detection of Botulinum neurotoxin, where steric hindrance is applied to achieve specific signal amplification via conformational change of the aptamer. The incubation time and potassium concentration of the reaction buffer were found to be key parameters affecting the sensitivity of detection of the recognition of Botulinum neurotoxin by the aptamer. Under optimized experimental conditions, a high signal-to-noise ratio was obtained within 24 h with a limit of detection (LOD) of 40 pg/ml by two standard deviation cutoffs above the noise level.     

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.     

Label-free aptasensor for platelet-derived growth factor (PDGF) protein

A label-free aptasensor for platelet-derived growth factor (PDGF) protein is reported. The aptasensor uses mixed self-assembled monolayers (SAMs) composed of a thiol-modified PDGF binding aptamer and 6-mercaptohexanol (MCH) on a gold electrode. The SAMs were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) before and after binding of the protein using [Fe(CN)₆]^3−/4−, a redox marker ion as an indicator for the formation of a protein-aptamer complex. The CVs at the PDGF modified electrode showed significant differences, such as changes in the peak currents and peak-to-peak separation, before and after binding of the target protein. The EIS spectra, in the form of Nyquist plots, were analyzed with a Randles circuit while the electron transfer resistance R_ct was used to monitor the binding of the target protein. The results showed that, without any modification to the aptamer, the target protein can be recognized effectively at the PDGF binding aptamer SAMs at the electrode surface. Control experiments using non-binding oligonucleotides assembled at the electrode surfaces also confirmed the results and showed that there was no formation of an aptamer-protein complex. The DPV signal at the aptamer functionalized electrode showed a linearly decreased marker ion peak current in a protein concentrations range of 1-40 nM. Thus, label-free detection of PDGF protein at an aptamer modified electrode has been demonstrated.     

Evaluation of Relative Aptamer Binding to Campylobacter jejuni Bacteria Using Affinity Probe Capillary Electrophoresis

Abstract

A qualitative capillary electrophoresis immunoassay was developed for the first-time to evaluate aptamer binding to bacterial cells. Binding affinity of aptamers developed against a Campylobacter jejuni bacterial cell target, relative to other common food-borne pathogens was investigated and specific binding affinity was evidenced by pronounced mobility shift and peak broadening with increasing bacteria concentration for both aptamers. Little to no mobility shift was observed for food-borne pathogens, Salmonella typhirium and Escherichia coli, even when increasing concentrations 10-fold over target. These results suggest that affinity probe capillary electrophoresis could be useful for qualitative screening of aptamer candidates for bacterial cell targets.     

Viscosity from ternary mixtures of two weakly charged polyelectrolytes in solution

The mode-mode coupling approximation (MMCA) is applied to describe the behavior of the reduced viscosity η_r from ternary mixtures of two weakly charged polyelectrolytes in solution. Similarly (ε = +1) as well as oppositely (ε = −1) weakly charged polyions are discussed in this work. The variation of η_r with the total polyelectrolyte concentration C_T exhibits a peak. The position and the height of this peak show for a given concentration of added salt C_S a sensitive variation with: the charge parameters (degrees of ionization) f₁ and f₂ of both polyelectrolytes, the proportion of polyelectrolyte 1 within the mixture (x = C₁/C_T) and the Flory interaction parameter χ_F between monomeric units of different species. This calculation is presented within the framework of the Rouse model where no hydrodynamic interactions are taken into account.     

Binding kinetics of human cellular prion detection by DNA aptamers immobilized on a conducting polypyrrole

We developed a biosensor based on the surface plasmon resonance (SPR) method for the study of the binding kinetics and detection of human cellular prions (PrP^C) using DNA aptamers as bioreceptors. The biosensor was formed by immobilization of various biotinylated DNA aptamers on a surface of conducting polypyrrole modified by streptavidin. We demonstrated that PrP^C interaction with DNA aptamers could be followed by measuring the variation of the resonance angle. This was studied using DNA aptamers of various configurations, including conventional single-stranded aptamers that contained a rigid double-stranded supporting part and aptamer dimers containing two binding sites. The kinetic constants determined by the SPR method suggest strong interaction of PrP^C with various DNA aptamers depending on their configuration. SPR aptasensors have a high selectivity to PrP^C and were regenerable by a brief wash in 0.1 M NaOH. The best limit of detection (4 nM) has been achieved with this biosensor based on DNA aptamers with one binding site but containing a double-stranded supporting part.

Aptameric SERS sensor for Hg~(2+) analysis using silver nanoparticles

Aptamer–silver nanoparticles (AgNPs) based surface-enhanced Raman scattering (SERS) sensor has been developed for Hg²⁺ detection by employing the structure-switching aptamer in the presence of spermine. This simple method shows excellent sensitivity and selectivity owing to the sensitive SERS detection technique and high specificity of aptamer for binding Hg²⁺.     

Detection of thrombin using an excimer aptamer switch labeled with dual pyrene molecules

We constructed an excimer aptamer probe containing one pyrene molecule at each end of a DNA aptamer to achieve the detection of thrombin, which binds to the heparin-binding site of thrombin with high binding affinity. The specific binding of thrombin to the excimer aptamer probe brought the two pyrene molecules at the termini of the duplex of the aptamer into close proximity, generating an excimer. The excimer emitted a distinct fluorescence peak, and fluorometric measurement of excimer allowed the sensitive detection of thrombin. The effects of experimental conditions like pH, ionic strength, and cations were investigated and optimized. The detection limit for thrombin was about 42 pM. This aptamer switch has potential in the study of molecular interactions and protein sensing with other switch-based detection strategy.

Intercalation of Proflavine in ssDNA Aptamers: Effect on Binding of the Specific Target Chloramphenicol

The structural modification of ssDNA‐based aptamers upon specific binding of its target molecule leads to changes of the charge‐transfer resistance (R_ct) of a negatively‐charged free‐diffusing redox probe. The aptamer adopts a structure due to self‐hybridization which is stabilized using profalvine as intercalator. The pre‐organized aptamer structure is used to detect chloramphenicol (CAP) requiring a substantial change of the aptamer structure indicated by a CAP concentration dependent increase in the R_ct values. Pre‐incubation of the aptamer‐modified electrode with an intercalator allows for the modulation of the aptamer/target interaction and hence for a modulation of the CAP‐dependent variation of the R_ct values.     

Development of a filtration-based SERS mapping platform for specific screening of Salmonella enterica serovar Enteritidis

The presence of Salmonella in natural freshwater and drinking water is a leading cause of intestinal illness all over the world; thus, the detection of Salmonella in water is of great importance to public health. The objective of this study is to develop a rapid screening method for the detection of Salmonella enterica serovar Enteritidis in water involving surface-enhanced Raman spectroscopy (SERS), aptamers, and filtration. SERS offers a great alternative to traditional methods of pathogen detection, with a simplified detection assay and shortened detection time. The specific capturing and labeling of Salmonella Enteritidis are realized by a specific single-stranded DNA aptamer, which is modified with an additional chain of adenine and fluorescein (FAM) and used as presence/absence indicator of Salmonella Enteritidis. By incorporating a vacuum filtration system, bacterial cells recognized by the specific aptamer are concentrated onto a membrane. With additional filtration of gold nanoparticles, the aptamer signals were captured and used to construct a SERS mapping indicating the presence and absence of target bacterial strains with potential quantitative capability. The specificity of the method was validated by using other strains of bacteria such as Escherichia coli and Listeria monocytogenes. The sensitivity of the method goes down to 10³ CFU/mL for 1 mL of sample with a total detection and analyzing time within 3 h. This study demonstrates the capability of the filtration-based SERS platform for detecting Salmonella Enteritidis in various aqueous matrices such as distilled water and rinsing water from fresh produce with high selectivity and sensitivity.

Graphical abstract

     

Novel extraction supports based on immobilised aptamers: evaluation for the selective extraction of cocaine

A new kind of selective sorbent based on the use of aptamers and dedicated to the selective solid phase extraction was developed. Cocaine aptamer was chosen as model aptamer to demonstrate the feasibility of this material and to provide a complete evaluation of the synthesized sorbent. The effect of different parameters such as the nature of the immobilisation support (silica, agarose), the type of immobilisation (covalent or non-covalent) and the length of the spacer arm (C₆ or C₁₂) were studied. Therefore, various oligosorbents based on different immobilisation strategies were synthesized and characterised by estimating the extraction recovery and the capacity of cocaine and the binding efficiency of aptamers. Control supports without immobilised aptamers were simultaneously studied in parallel to assess the selectivity brought by the oligosorbents. The oligosorbent based on CNBr-activated sepharose showed the best performances with an extraction recovery for cocaine of 90% while 6% was obtained on the control sorbent. The high selectivity brought by the oligosorbent was then illustrated by applying the oligoextraction followed by LC/MS analysis to a post-mortem blood (cocaine overdose). Results were compared to those resulting from a conventional protein precipitation procedure. The presence of co-extracted interfering compounds was strongly reduced with the treatment by oligoextraction. A limit of quantification of 0.5 ng/mL was obtained that is largely lower than the concentration found after a single intake of cocaine.     

A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

Sensitive and selective detection of Pb²⁺ is of great importance to both human health and environmental protection. Here we propose a novel fluorescence anisotropy (FA) approach for sensing Pb²⁺ in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb²⁺. It was found that the aptamer probe had a high FA in the absence of Pb²⁺. This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb²⁺ to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb²⁺. Indeed, we observed a decrease in FA of aptamer probe upon Pb²⁺ binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb²⁺. Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb²⁺ in homogeneous solution. The change in FA showed a linear response to Pb²⁺ from 10 nM to 2.0 μM, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb²⁺ over other metal ions. The method is simple, quick and inherits the advantages of aptamer and FA.