Aptamer‐modified magnetic metal‐organic framework MIL‐101 for highly efficient and selective enrichment of ochratoxin A

A facile and efficient strategy is developed to modify aptamers on the surface of the magnetic metal‐organic framework MIL‐101 for the rapid magnetic solid‐phase extraction of ochratoxin A. To the best of our knowledge, this is the first attempt to create a robust aptamer‐modified magnetic MIL‐101 with covalent bonding for the magnetic separation and enrichment of ochratoxin A. The saturated adsorption of ochratoxin A by aptamer‐modified magnetic MIL‐101 was 7.9 times greater than that by magnetic metal‐organic framework MIL‐101 due to the former's high selective recognition as well as good stability. It could be used for extraction more than 12 times with no significant changes in the extraction efficiency. An aptamer‐modified magnetic MIL‐101‐based method of magnetic solid‐phase extraction combined with ultra high performance liquid chromatography with tandem mass spectrometry was developed for the determination of trace ochratoxin A with limit of detection of 0.067 ng/L. Ochratoxin A of 4.53–13.7 ng/kg was determined in corn and peanut samples. The recoveries were in the range 82.8–108% with a relative standard deviation (n = 5) of 4.5–6.5%. These results show that aptamer‐modified magnetic MIL‐101 exhibits selective and effective enrichment performance and have excellent potential for the analysis of ultra‐trace targets from complex matrices.     

Label‐free Electrochemical Aptasensor for Carcino‐embryonic Antigen Based on Ternary Nanocomposite of Gold Nanoparticles,Hemin and Graphene

In this report, a label‐free electrochemical aptasensor for carcino‐embryonic antigen (CEA) was successfully developed based on a ternary nanocomposite of gold nanoparticles, hemin and graphene nanosheets (AuNPs‐HGNs). This nanocomposite was prepared by decorating gold nanoparticles on the surface of hemin functionalized graphene nanosheets via a simple wet‐chemical strategy. The aptamer can be assembled on the surface of AuNPs‐HGNs/GCE (glassy carbon electrode) through Au‐S covalent bond to form the sensing interface. Hemin absorbed on the graphene nanosheets not only acts as a protective agent of graphene sheets, but also as an in situ probe base on its excellent redox properties. Gold nanoparticles provide with both numerous binding sites for loading CEA binding aptamer (CBA) and good conductivity to promote the electron transfer. The current changes, which are caused by CEA specifically binding on the modified electrode, are exploited for the label‐free detection of CEA in a very rapid and convenient protocol. Therefore, the method has advantages of high sensitivity, wide linear range (0.0001–10 ng mL^?1), low detection limit (40 fg mL^?1) and attractive specificity. The results illustrate that the proposed label‐free electrochemical aptasensor has a potential application in the biological or clinical target analysis for its simple operation and low cost.     

Amperometric sandwich-type aptasensor based on peroxidase mimetic Au@Pt for the carcinoembryonic antigen

For sensitive analysis of cancer biomarker carcinoembryonic antigen (CEA), an amperometric sandwich-type aptasensor is proposed based on a signal amplification strategy of Au@Pt bimetallic nanoprobes. As the excellent catalytic activity to hydrogen peroxide (H₂O₂), core-shell Au@Pt nanoparticles are employed as nanoprobes by conjugating directly with the secondary aptamer of CEA (Apt-II). Due to the synergic recognition effect of dual aptamers and the excellent catalytic activity of nanoprobes, this amperometric sandwich-type aptasensor for CEA exhibits high specificity and good sensitivity with a limit of detection of 0.31 ng/mL, along with a wide linear range from 0.1 ng/mL to 100 ng/mL.     

Preparation of aptamer‐bound polyamine affinity monolithic column via a facile triazine‐bridged strategy and application to on‐column specific discrimination of ochratoxin A

Developing a high‐performance modification protocol is critical for efficiently fabricating affinity monolith. Herein, by using 2,4,6‐trichloro‐1,3,5‐triazine as the linker, a simple triazine‐bridged approach was proposed for efficiently fabricating aptamer‐grafted polyhedral oligomeric silsesquioxane‐polyethyleneimine affinity monolith with high specificity toward ochratoxin A. Experimental parameters, column characteristics and specificity performances of the resultant affinity monolith were investigated in detail. Under the optimal conditions, 2,4,6‐trichloro‐1,3,5‐triazine was rapidly grafted on the polyamine matrix, and effectively applied to the subsequent bridge linkage of aptamers. It was simple and effective, which resulted in a significant decrease of modification time, excellent properties including the high coverage density of aptamer up to 1799 pmol/μL and sensitive detection of ochratoxin A as low as 10 pg/mL in beer samples. This protocol provides a facile approach for fabricating aptamer‐grafted polyamine affinity monoliths with highly selective discrimination performance.     

Simple and Fast Picomolar Detection of Ochratoxin A Using a Reusable Label Free Aptasensor Built with a Layer‐by‐layer Procedure

A label free impedimetric aptasensor for simple, fast and reusable picomolar detections of Ochratoxin A (OTA) in grape juices was designed. Two main factors were observed to affect the accurate detections of the toxin: i‐lateral interactions between self‐assembled aptamers ii‐ adsorption of large molecules present in complex matrixes like grape juices. Lateral interactions between aptamers were minimized by constructing the aptasensor in a Layer‐by‐Layer procedure. The interferences associated to the unspecific and irreversible adsorption of large molecules present in grape juice, were reduced by submitting samples to ultrafiltration prior to analysis. With this protocol, a 0.12 pM limit of detection and 0.24 pM limit of quantification in spiked grape juices were achieved after only 5–7 mins of interaction with the samples. The presented aptasensor can be recovered after a simple immersion in hot water (90 °C) for ten minutes.     

Particle Display: A Quantitative Screening Method for Generating High‐Affinity Aptamers

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution‐phase aptamers into “aptamer particles”, each displaying many copies of a single sequence on its surface. We then use fluorescence‐activated cell sorting (FACS) to individually measure the relative affinities of >10⁸ aptamer particles and sort them in a high‐throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high‐affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high‐quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.     

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.     

Label‐Free Electrochemical Aptasensor for Determination of Chloramphenicol Based on Gold Nanocubes‐Modified Screen‐Printed Gold Electrode

An ultrasensitive label‐free electrochemical aptasensor was developed for selective detection of chloramphenicol (CAP). The aptasensor was made using screen‐printed gold electrode modified with synthesized gold nanocube/cysteine. The interactions of CAP with aptamer were studied by cyclic voltammetry, square wave voltammetry (SWV) and electrochemical impedance spectroscopy. Under optimized conditions, two linear calibration curves were obtained for CAP determination using SWV technique, from 0.03 to 0.10 µM and 0.25–6.0 µM with a detection limit of 4.0 nM. The aptasensor has the advantages of good selectivity and stability and applied to the determination of CAP in human blood serum sample.     

Aptamer sandwich assays: human α-thrombin detection using liposome enhancement

Fluorescent dye-encapsulating liposomes tagged with aptamers were developed and used as reporting signals in an aptamer-based sandwich assay. α-Thrombin was utilized as a prototypical analyte as two well-studied aptamers binding distinct epitopes are available to form a sandwich complex. Cholesteryl–TEG-modified aptamers were embedded into the liposomal lipid bilayer while the interior cavity of the liposomes encapsulated fluorescent sulforhodamine B dye. Such liposomes successfully formed a sandwich complex with α-thrombin and a microtiter plate immobilized aptamer, proving that aptamers retain their ability to fold when anchored to the liposome surface. Parameters studied included liposomal aptamer coverage, sandwich aptamer orientation, aptamer label orientation, aptamer spacer length and type, incubation buffer, and aptamer concentration. The optimized conditions found here in the fluorescence assay led to a limit of detection of 64 pM or 2.35 ng/mL, corresponding to 6.4 fmol or 235 pg, respectively, in a 100 μL volume. This is an order of magnitude lower than previous sandwich aptamer assays using the same sequences with lowest reported limits of detection of 0.45 nM. In addition, the assay was applied successfully to the detection of α-thrombin in human plasma. The success of this method in a standard microtiter plate format and the relatively facile functionalization of liposomes with aptamers suggest that this approach provides a versatile option for routine analytical applications.     

Sandwich-type aptasensor employing modified aptamers and enzyme-DNA binding protein conjugates

The use of aptamers in various analytical applications as molecular recognition elements and alternative to antibodies has led to the development of various platforms that facilitate the sensitive and specific detection of targets ranging from small molecules and proteins to whole cells. The goal of this work was to design a universal and adaptable sandwich-type aptasensor exploiting the unique properties of DNA binding proteins. Specifically, two different enzyme-DNA binding protein conjugates, GOx-dHP and HRP-scCro, were used for the direct detection of a protein using two aptamers for target capture and detection. The specific dsDNA binding sequence for each DNA binding protein tag was incorporated in the form of a hairpin at one end of each aptamer sequence during the synthesis step. Detection was accomplished by an enzymatic (GOx/HRP) cascade reaction after the binding of each enzyme conjugate to its corresponding binding sequence on each aptamer. The proposed sandwich-type aptasensor was validated for the detection of thrombin, which is one of the most commonly used model targets with known dual aptamers. The limit of detection accomplished was 0.92 nM which is comparable with other colorimetric platforms reported in the literature. The sensitivity of the aptasensor was easily modulated by changing the number of dsDNA binding sites incorporated in the aptamer sequences, thus controlling the enzyme stoichiometry. Finally, the potential use of the proposed sensing approach for real sample testing was demonstrated using spiked human plasma and no significant matrix effects were observed when up to 2% plasma was used.

     

Sensor Platform with a Custom‐tailored Aptamer for Diagnosis of Synthetic Cannabinoids

Synthetic cannabinoids (SCs) are the large group of abused drugs and detection of them is still a challenge. Hence, new methods for analysis of SCs are being investigated. We aimed to develop a novel system for selective analysis of SCs. First, various custom‐tailored aptamers against the target SCs were selected through GO‐SELEX process. Toggling between different SC analytes during successive rounds of selection was performed to generate cross‐reactive aptamers. Then, the amino‐capped aptamers were synthesized and easily attached to the cysteamine‐covered gold electrodes. Analytical parameters and selectivity of the aptasensors were compared by using electrochemical techniques. After comparison of the analytical features and selectivity towards target analytes, one of the aptamers designated as Apta‐1 was chosen for further measurements. The aptasensor was tested by using differential pulse voltammetry technique against JWH‐018 (5‐pentanoic acid), selected as a model for SCs. The linearity and limit of detection were determined as 0.01–1.0 ng/mL and 0.036 ng/mL. Finally, sample application in synthetic urine samples was successfully performed with standard addition method, as confirmed by LC‐QTOF/MS. JWH‐018 (4‐hydroxypentyl), JWH‐073 (3‐hydroxybutyl), JWH‐250 (5‐hidroxypentyl) and HU‐210 were used to test the selectivity of the aptasensor and the system was shown to recognize all these SCs. Also other illegal drugs did not significantly interfere with the signal responses.     

Aptasensor for ampicillin using gold nanoparticle based dual fluorescence–colorimetric methods

A gold nanoparticle based dual fluorescence–colorimetric method was developed as an aptasensor to detect ampicillin using its single-stranded DNA (ssDNA) aptamer, which was discovered by a magnetic bead-based SELEX technique. The selected aptamers, AMP4 (5′-CACGGCATGGTGGGCGTCGTG-3′), AMP17 (5′-GCGGGCGGTTGTATAGCGG-3′), and AMP18 (5′-TTAGTTGGGGTTCAGTTGG-3′), were confirmed to have high sensitivity and specificity to ampicillin (K _d, AMP7 = 9.4 nM, AMP17 = 13.4 nM, and AMP18 = 9.8 nM, respectively). The 5′-fluorescein amidite (FAM)-modified aptamer was used as a dual probe for observing fluorescence differences and color changes simultaneously. The lower limits of detection for this dual method were a 2 ng/mL by fluorescence and a 10 ng/mL by colorimetry for ampicillin in the milk as well as in distilled water. Because these detection limits were below the maximum residue limit of ampicillin, this aptasensor was sensitive enough to detect antibiotics in food products, such as milk and animal tissues. In addition, this dual aptasensor will be a more accurate method for antibiotics in food products as it concurrently uses two detection methods: fluorescence and colorimetry.     

Selection of cholesterol esterase aptamers using a dual‐partitioning approach

Non‐systematic evolution of ligands by exponential enrichment and other capillary‐based methods have grown in popularity for selection of aptamers since they provide a fast and efficient partitioning method when compared to classical techniques. Despite promising developments in these techniques, a major obstacle needs to be overcome for capillary‐based selections to be widely accepted. Due to the small injection volumes associated with CE, only a small proportion of the nucleic acid library can be partitioned at any one time. In this paper, we propose a new two‐step method for the selection of aptamers which firstly incorporates a nitrocellulose membrane filter followed by CE. This technique allows for nonbinding sequences to be eliminated, reducing the library size before subsequent capillary‐based partitioning, while still reducing the time taken for aptamers to be selected. We demonstrated this technique on the selection of aptamers for cholesterol esterase and the highest binding truncated aptamer CES 4T displayed a K_D of 203 ± 14 nM. In addition, an increase in the number of sequences partitioned was estimated using spectrophotometry and capillary injection volumes. The results suggested that for successful selection a two‐step approach is needed. This hybrid technique could be used to select aptamers that bind to targets both in solution and immobilized onto a stationary phase, allowing the aptamers to be used in different binding environments.     

Label‐Free Electrochemical Thrombin Aptasensor Based on Ag Nanoparticles Modified Electrode

A novel label‐free electrochemical method for protein detection based on redox properties of silver was developed. As recognition elements, thrombin‐binding aptamers were used. Screen printed electrodes modified with silver nanoparticles (AgNP) were employed as a sensing platform for aptasensor devices. The oxidation of silver upon polarization served as a basis for analytical response. Three different thrombin binding aptamers with various surface concentrations were studied. Linear range of aptasensor response corresponded to the 10⁻⁹ M to 10⁻⁷ M thrombin concentration range and the detection limit was 10⁻⁹ M.     

Selection of a DNA aptamer for the development of fluorescent aptasensor for carbaryl detection

An improved ssDNA library immobilized systematic evolution of ligands by enrichment(SELEX) was applied to select aptamers against carbaryl.After nine selection rounds,a highly enriched ssDNA pool was obtained.The Apta3 was demonstrated as the optimal aptame r.In order to facilitate the modification of aptamer,the Apta3 was further truncated with the dissociation constant(K_d) of 0.3 64 ± 0.055 μmol/L and a fluorescent aptasensor was developed.The linear range for carbaryl was from 100 nmol/L to1500 nmol/L,with the limit of detection was as low as 15.23 nmol/L.Besides,the biosensor was validated for the carbaryl spiked real samples,and the recoveries were between 97.7% and 107.3%.     

Direct detection of OTA by impedimetric aptasensor based on modified polypyrrole-dendrimers

Ochratoxin A (OTA) is a carcinogenic mycotoxin that contaminates food such as cereals, wine and beer; therefore it represents a risk for human health. Consequently, the allowed concentration of OTA in food is regulated by governmental organizations and its detection is of major agronomical interest. In the current study we report the development of an electrochemical aptasensor able to directly detect trace OTA without any amplification procedure. This aptasensor was constructed by coating the surface of a gold electrode with a film layer of modified polypyrrole (PPy), which was thereafter covalently bound to polyamidoamine dendrimers of the fourth generation (PAMAM G4). Finally, DNA aptamers that specifically binds OTA were covalently bound to the PAMAM G4 providing the aptasensor, which was characterized by using both Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) techniques. The study of OTA detection by the constructed electrochemical aptasensor was performed using Electrochemical Impedance Spectroscopy (EIS) and revealed that the presence of OTA led to the modification of the electrical properties of the PPy layer. These modifications could be assigned to conformational changes in the folding of the aptamers upon specific binding of OTA. The aptasensor had a dynamic range of up to 5 μg L⁻¹ of OTA and a detection limit of 2 ng L⁻¹ of OTA, which is below the OTA concentration allowed in food by the European regulations. The efficient detection of OTA by this electrochemical aptasensor provides an unforeseen platform that could be used for the detection of various small molecules through specific aptamer association.     

Label-free electrochemical aptasensor for the detection of lysozyme

This work reports the advantages of a label free electrochemical aptasensor for the detection of lysozyme. The biorecognition platform was obtained by the adsorption of the aptamer on the surface of a carbon paste electrode (CPE) previously blocked with mouse immunoglobulin under controlled-potential conditions. The recognition event was detected from the decrease in the guanine and adenine electro-oxidation signals produced as a consequence of the molecular interaction between the aptamer and lysozyme. The biosensing platform demonstrated to be highly selective even in the presence of large excess (9-fold) of bovine serum albumin, cytochrome C and myoglobin. The reproducibility for 10 repetitive determinations of 10.0 mg L⁻¹ lysozyme solution was 5.1% and 6.8% for guanine and adenine electro-oxidation signals, respectively. The detection limits of the aptasensor were 36.0 nmol L⁻¹ (if considering guanine signal) and 18.0 nmol L⁻¹ (if taking adenine oxidation current). This new sensing approach represents an interesting and promising alternative for the electrochemical quantification of lysozyme.     

A Dendrimer Supported Electrochemical Immunosensor for the Detection of Alpha‐feto protein – a Cancer Biomarker

The electrochemical detection of alpha‐feto protein based on novel gold nanoparticles‐ poly(propylene imine) dendrimer platform is reported. The platform was prepared by co‐electrodeposition of gold nanoparticles and generation 3 poly (propylene imine) dendrimer on a glassy carbon electrode. Each modifying step was characterised by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical measurements showed that the platform was stable, conducting and exhibited reversible electrochemistry. Results obtained from the electrochemical impedance spectroscopy interrogation in [Fe(CN)₆^3−/4−] redox probe showed a marked reduction in charge transfer resistance (R_ct) after each modification step. The immunosensor was prepared by immobilisation of a probe anti‐alpha feto protein (AFP) on the platform for 3 hrs at 35 °C followed by blocking the surface with bovine serum albumin to minimise non‐specific binding. The prepared immunosensor was used to detect AFP over a wide concentration range from 0.005 to 500 ng/mL and detection limits of 0.0022 and 0.00185 ng/mL were obtained for SWV and EIS measurements respectively. The immunosensor gave good stability over a period of fourteen days when stored at 4 °C.     

Finding the Optimal Surface Density of Aptamer Monolayers by SPR Imaging Detection‐based Aptamer Microarrays

Surface plasmon resonance imaging (SPRi) by enabling label‐free, real time assessment of biomolecular interactions in multiplexed manner is one of the methods of choice for high throughput characterization of large pools of DNA aptamer candidates following in vitro selection. Moreover, with major advances in in situ amplification methods SPRi became also a viable detection platform for aptamer microarrays. In case of aptamer microarrays, commonly prepared by microspotting, the direct assessment of the surface density of aptamer probes, which is essential for both kinetic and sensing measurements is not possible. Therefore, here we introduce a methodology for simple, one‐step determination of surface densities of thiol labelled aptamer monolayers microspotted on gold SPRi chips. Based on this methodology we investigated in detail the effect of the surface density of aptamers on target binding through two aptamer‐target systems, i. e. human immunoglobulin E (hIgE) and six histidine tag 6xHis‐tag. We found that the surface density of the aptamers is indeed critical and shows a sharp maximum in terms of target binding efficiency, which is largely determined by the size of the target. The optimal aptamer surface densities determined, the immobilization chemistry (shared by many detection platforms, e. g., electrochemical, surface acoustic) and the trends identified may be used for rapid rational optimization of aptamer‐target assays.