Tris buffer-accelerated ligand exchange rate for instant fluorescence detection of trivalent chromium ion.

Functional nucleic acids (FNAs) have attracted a lot of attention for the rapid detection of metal ions. Cr3+ is one of the major heavy metal ions in natural waters. Due to the slow ligand exchange rate of Cr3+, the FNA-based Cr3+ sensors require long assay times, limiting the on-site applications. In this study, we report that the good's buffers containing amino and polyhydroxy groups greatly increase the ligand exchange rate of Cr3+. Using EDTA as a model coordinate ligand, the Tris buffer (100 mM, pH 7.0) showed the best acceleration effect among the eight buffers. It improved the rate constant ∼20-fold, shorten the half-time 19-fold, and lowered the activation energy ∼70% at 40 °C. The Tris buffer was then applied for sensor based on the Cr3+-binding induced fluorescence quenching of fluorescein (FAM)-labeled and single-stranded DNA (ssDNA), which shortened the assay time from 1 h to 1 min. The Tris buffer also ∼100% enhanced the fluorescence intensity of FAM, achieving the 11.4-fold lower limit of detection (LOD = 6.97 nM, S/N = 3). By the combination use of the Tris buffer and ascorbic acid, the strong interference from Cu2+, Pb2+, and Fe3+ suffered in many previous reported Cr3+ sensors was avoided. The practical application of the sensor for the detection of Cr3+ spiked in the real water samples were demonstrated with high recovery percentages. The Tris buffer could be applied for other metal ions with slow ligand exchange rate (such as V2+, Co3+ and Fe2+) to solve diverse issues such as long assay time and low synthesis yield of metal complexes, without the need of heating treatment.

Selection of regioselective DNA aptamer for detection of homocysteine in nondeproteinized human plasma.

Small molecule-binding aptamers often suffer from high cross reactivity to structure analogues in biological samples, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by performing binding-inhibited organic reaction-based regioselective selection of aptamers against homocysteine (Hcy), which is a marker for diagnosing many disorders including stroke and Alzheimer's. This approach has led to isolation of a DNA aptamer that binds to the alkane thiol chain of Hcy with exceptional specificity against cysteine. It also binds with oxidized Hcy at weaker affinity. Using this new aptamer, we produced a reusable fluorescent optical fiber aptasensor for direct and validated detection of both free and total Hcy in nondeproteinized patient plasma in the diagnostic concentration range. The binding site-specific aptamer selection and optical-fiber-sensing strategy can expand the practical utility of aptamers in clinical diagnosis.

Recent Progress in Functional-Nucleic-Acid-Based Fluorescent Fiber-Optic Evanescent Wave Biosensors.

Biosensors capable of onsite and continuous detection of environmental and food pollutants and biomarkers are highly desired, but only a few sensing platforms meet the "2-SAR" requirements (sensitivity, specificity, affordability, automation, rapidity, and reusability). A fiber optic evanescent wave (FOEW) sensor is an attractive type of portable device that has the advantages of high sensitivity, low cost, good reusability, and long-term stability. By utilizing functional nucleic acids (FNAs) such as aptamers, DNAzymes, and rational designed nucleic acid probes as specific recognition ligands, the FOEW sensor has been demonstrated to be a general sensing platform for the onsite and continuous detection of various targets ranging from small molecules and heavy metal ions to proteins, nucleic acids, and pathogens. In this review, we cover the progress of the fluorescent FNA-based FOEW biosensor since its first report in 1995. We focus on the chemical modification of the optical fiber and the sensing mechanisms for the five above-mentioned types of targets. The challenges and prospects on the isolation of high-quality aptamers, reagent-free detection, long-term stability under application conditions, and high throughput are also included in this review to highlight the future trends for the development of FOEW biosensors capable of onsite and continuous detection.

Ultrasensitive evanescent wave optical fiber aptasensor for online, continuous, type-specific detection of sulfonamides in environmental water.

Sensors capable for online continuous monitoring of total sulfonamides in environmental waters are highly desired due to their adverse effects on ecosystem, unexpected concentration fluctuation, and diversity. At present, no sensor with this capability has been reported. In this study, we evaluated the cross reactivity (CR) of the previously reported sulfadimethoxine-binding aptamer using DNase I assay and found that the aptamer was type-specific to sulfonamides. We then fabricated the first type-specific sulfonamide sensor, where the aptamer was immobilized on the optical fiber of the evanescent wave sensor, followed by the surface coating with Tween 80. The competitive binding of sulfonamides and Cy5.5 labeled complementary DNA enabled the low femtomolar to picomolar sensitivity and the detection of total 14 sulfonamides spiked in the lake water. The sensor also exhibited high selectivity, regeneration capability (40 cycles), stability (65 days), and short detection time (5 min). In addition, we found that the CRs were greatly dependent on the buffer composition. By performing the parallel detections in two buffers, the sensors detected 18 out of the 24 sulfonamides with the diversity coverage higher than commercial ELISA kits. Our aptasensor fills the technical gap for continuous monitoring of total sulfonamides in environmental waters.

Evanescent Wave Optical-Fiber Aptasensor for Rapid Detection of Zearalenone in Corn with Unprecedented Sensitivity.

Zearalenone (ZEN) is a common mycotoxin pollutant found in agricultural products. Aptamers are attractive recognition biomolecules for the development of mycotoxin biosensors. Even though numerous aptasensors have been reported for the detection of ZEN in recent years, many of them suffer from problems including low sensitivity, low specificity, tedious experimental steps, high-cost, and difficulty of automation. We report here the first evanescent wave optical-fiber aptasensor for the detection of ZEN with unprecedented sensitivity, high specificity, low cost, and easy of automation. In our aptasensor, a 40-nt ZEN-specific aptamer (8Z31) is covalently immobilized on the fiber. The 17-nt fluorophore Cy5.5-labeled complementary DNA strand and ZEN competitively bind with the aptamer immobilized on the fiber, enabling the signal-off fluorescent detection of ZEN. The coating of Tween 80 enhanced both the sensitivity and the reproducibility of the aptasensor. The sensor was able to detect ZEN spiked-in the corn flour extract with a semilog linear detection range of 10 pM-10 nM and a limit of detection (LOD, S/N = 3) of 18.4 ± 4.0 pM (equivalent to 29.3 ± 6.4 ng/kg). The LOD is more than 1000-fold lower than the maximum ZEN residue limits set by China (60 µg/kg) and EU (20 µg/kg). The sensor also has extremely high specificity and showed negligible cross-reactivity to other common mycotoxins. In addition, the sensor was able to be regenerated for 28 times, further decreasing its cost. Our sensor holds great potential for practical applications according to its multiple compelling features.

Shortened and multivalent aptamers for ultrasensitive and rapid detection of alternariol in wheat using optical waveguide sensors.

Alternariol (AOH) is one of the common mycotoxins existing in a variety of foods at low level. Aptamers hold great promise for the development of sensitive and rapid aptasensors, but suffer from the excessive length and the difficulty in identification of critical binding domains (CBDs). In this study, the 5 nt CBD of the original 59-nt AOH aptamer (AOH-59, KD = 423 nM) was identified to be a 'C' bulge in between two A-T base pairs. AOH-59 was successfully shortened to a 23 nt aptamer (AOH 6C, KD = 701 nM). A 30 nt bivalent aptamer B-2-3 (KD = 445 nM) and a 39 nt trivalent aptamer T-2-3 (KD = 274 nM) were obtained by simply incorporating one or two CBDs into AOH 6C. The AOH 6C-, B-2-3-, and T-2-3-based optical waveguide aptasensors possessed the unprecedented detection of limits (LODs, S/N = 3) of 42 ± 3, 6 ± 1 and 2 ± 1 fM, respectively. Using the AOH 6C-based sensor as an example, we further demonstrated the detection of AOH spiked in wheat powder with a LOD of 37 pg/g, 20-230-fold lower than those achieved by ELISAs. The sensor was capable for 35 times 2-min regeneration and the assay time including the extraction of AOH from wheat was only about 1 h. We not only devised the first aptasensors for AOH detection, but also provided a simple strategy to design multivalent aptamers for small molecule targets.

A highly specific aptamer probe targeting PD-L1 in tumor tissue sections: Mutation favors specificity.

Although DNA aptamers can show comparable affinity to antibodies and have the advantage of having high batch-to-batch consistency, they often suffer from unsatisfied specificity for complex samples. The limited library size used for aptamer in vitro isolation (SELEX) has been recognized as one of the major reasons. Programmed cell death-ligand 1 (PD-L1) is both a key protein in cancer diagnostics and also immunotherapy. We report here a DNA aptamer that highly specifically binds PD-L1 expressed on the surface of various cancer cells and multiple types of tissue sections. The aptamers were selected from a DNA library containing a type II restriction endonuclease Alu I recognition site in the middle of the 40-nt random sequences, against recombinant PD-L1 rather than the whole cell or tissue section. The library enrichment was achieved by Alu I mediated-SELEX, named as REase-SELEX, in which Alu I cut off the non-binders at the recognition site and, more importantly, induced library mutations to substantially increase the library diversity. 8-60, a representative aptamer with high affinity (KD = 1.4 nM determined by SPR) successfully detected four types of cancer cells with PD-L1 expression levels from low to high by flow cytometry, normal human tonsil (gold standard for PD-L1 antibody evaluation), clinical non-small cell lung cancer (high PD-L1 expression level), and malignant melanoma (low PD-L1 expression level) tissue sections by fluorescence microscopy imaging, showing unprecedented high specificity. The results demonstrate that 8-60 is an advanced probe for PD-L1 cancer diagnostics and mutations in SELEX greatly favor aptamer specificity.

PD-L1 aptamer isolation via Modular-SELEX and its applications in cancer cell detection and tumor tissue section imaging.

PD-1/PD-L1 is an important pathway in immunotherapy and a high PD-L1 expression level in tumor tissues is an essential prerequisite for PD-1/PD-L1 blocking-based therapy. The PD-L1 expression level in tumor tissue sections is currently detected via immunohistochemistry (IHC) using anti-PD-L1 antibodies from various resources, which has the disadvantage of inconsistent results. As synthetic affinity ligands, aptamers have good batch-to-batch consistency and have been demonstrated to have great potential for use in biomedical applications. In this study, we isolated PD-L1 aptamers using a combination method, named Modular-SELEX (systematic evolution of ligands by exponential enrichment), which includes three sequentially performed modules: the affinity module, the specificity module, and the compatibility module. Three rounds of magnetic crosslinking precipitation (MCP)-SELEX, three rounds of Capture-SELEX, and two rounds of Tissue-SELEX were respectively performed in the corresponding three modules to significantly and efficiently improve the native affinity, specificity, and compatibility of the enriched library. The isolated aptamer Clon-3 had nanomolar binding affinity, as determined via both homogeneous and PD-L1 immobilized affinity assays. Clon-3 could be used to recognize various cancer cells with distinct PD-L1 expression levels using flow cytometry. The PD-L1 expression levels in normal human tonsils (the gold standard for anti-PD-L1 antibody) and non-small cell lung cancer tissue sections stained using Cy5.5-labeled Clon-3 were also successfully imaged using a confocal microscope. The fluorescence intensities of the tissue sections were in good agreement with their actual PD-L1 expression levels as confirmed via IHC.

Nanoscale Affinity Double Layer Overcomes the Poor Antimatrix Interference Capability of Aptamers.

Poor antimatrix interference capability of aptamers is one of the major obstacles preventing their wide applications for real-sample detections. Here, we devise a multiple-function interface, denoted as a nanoscale affinity double layer (NADL), to overcome this bottleneck via in situ simultaneous target enrichment, purification, and detection. The NADL consists of an upper aptamer layer for target purification and sensing and a lower nanoscale solid-phase microextraction (SPME) layer for sample enrichment. The targets flowing through the NADL-functionalized surface are instantly million-fold enriched and purified by the sequential extraction of aptamer and SPME. The formation of the aptamer-target complex is greatly enhanced, enabling ultrasensitive detection of targets with minimized interference from the matrix. Taking the fiber-optic evanescent wave sensor as an example, we demonstrated the feasibility and generality of the NADL. The unprecedented detection of limits of 800, 4.8, 40, and 0.14 fM were, respectively, achieved for three representative small-molecule targets with distinct hydrophobicity (kanamycin A, sulfadimethoxine, and di-(2-ethylhexyl) phthalate) and protein target (human serum albumin), corresponding to 2500 to 3 × 108-fold improvement compared to the sensors without the NADL. Our sensors also showed exceptionally high target specificity (>1000) and tunable dynamic ranges simply by manipulating the SPME layer. With these features comes the ability to directly detect targets in diluted environmental, food, and biological samples at concentrations all well below the tolerance limits.

Strong additive and synergistic effects of polyoxyethylene nonionic surfactant-assisted protein MALDI imaging mass spectrometry.

Protein MALDI imaging mass spectrometry (MALDI-IMS) holds a great promise to acquire spatial distribution information of proteins on biological tissue, but it suffers from the small number of proteins detected by direct MALDI-IMS detection. Ionic surfactants have been extensively used for protein extraction to improve the number of proteins detected in tissue samples by LC-MS analysis, but seldom by direct MALDI-IMS detection. Nonionic surfactants are milder than ionic surfactants and protein native structures are remained after extraction, which favors the spatial resolution of direct MALDI-IMS. However, nonionic surfactants are less effective than ionic surfactants. In this report, we utilized polyoxyethylene nonionic surfactants (PNS) to pre-incubate the tissue section, followed by the on-tissue trypsin digestion and then direct MALDI detection of in-situ formed peptides. For the first time, we observed that the additive effect of PNS and the synergistic effect of the mixed PNS in improving the number of peptides detected. Specifically, the peptides detected were 73.0-90.7% distinct when the different PNS (Tween 80 or Triton X-100 alone or their mixture) was used. Taking advantage of this additive effect, the 96 proteins including 12 transmembrane proteins were detected, corresponding to a ~10-fold improvement compared to MALDI-IMS without surfactant. When the mixed surfactants were used to replace Tween 80 and Triton X-100 alone, the optimized surfactant concentration decreased 20-100-fold and the number of peptides detected with m/z > 2500 Da was improved 15-fold. The additive and synergistic effects of PNS suggested that the interaction mode between each PNS and proteins is highly variable. Benefiting from the strong additive effect and diversity of PNS, further improvement of the number of proteins detected by MALDI-IMS is clearly feasible.

Nano-Affi: a solution-phase, label-free, colorimetric aptamer affinity assay based on binding-inhibited aggregation of gold nanoparticles.

The ideal way to assess aptamer affinity is when both aptamer and target are in a native state, without the unpredictable interference associated with labelling and surface immobilization. However, most current aptamer affinity assays need aptamer (or target) immobilization on surface and/or labelling. Ideally, such a solution-phase assay should also be high-throughput, in order to accelerate aptamer identification, binding site study, and engineering for various downstream applications. So far, only isothermal titration calorimetry (ITC) enables label-free solution-phase affinity measurements, but with low-throughput and the need of large amount of samples. Here, we report a solution-phase, label-free, colorimetric gold nanoparticle (AuNP)-based affinity assay (Nano-Affi) that addresses this need. Nano-Affi is based on kinetically-favoured, adsorbate charge-tuned aggregation of AuNPs, wherein positively-charged or near-neutral proteins induce instantaneous aggregation of negatively-charged AuNPs at the pH below or near the isoelectric point of the target protein. In contrast, protein-aptamer complexes possess a greater negative charge than free targets, and thus induce little or no aggregation of AuNPs due to electrostatic repulsion. The higher an aptamer's affinity for the protein, the less AuNP aggregation occurs. We demonstrate here that Nano-Affi enables the reliable aptamer screening and dissociation constant determination for diverse protein targets, as well as binding site identification, with readouts based on colour observation or absorbance or dynamic light scattering size measurements. Nano-Affi possesses sub-nanomolar sensitivity and can be performed with nanogram amounts of protein in less than half an hour with minimal training and minimal instrument requirements.

Universal Design of Structure-Switching Aptamers with Signal Reporting Functionality.

Structure-switching aptamers (SSAs) offer a promising recognition element for sensor development. However, the generation of SSAs via in vitro aptamer selection technologies or postselection engineering is challenging. Inspired by the two-domain structure of antibodies, we have devised a simple, universal strategy for engineering aptamers into SSAs with signal reporting functionality. These constructs consist of a "constant" domain, comprising a split DNAzyme G-quadruplex (G4) region for signal transduction, and a "variable" domain, comprising an aptamer sequence capable of specific target binding. In the absence of target, the G4-SSA construct folds into a parallel G4 structure with high peroxidase catalytic activity. Target binding disrupts the G4 structure, resulting in low enzymatic activity. We demonstrate that this change in DNAzyme activity enables sensitive and specific colorimetric detection of diverse targets including Hg2+, thrombin, sulfadimethoxine, cocaine, and 17ß-estradiol. G4-SSAs can also achieve label-free fluorescence detection of various targets using a specific G4-binding dye. We demonstrate that diverse aptamers can be readily engineered into G4-SSA constructs independent of target class, binding affinity, aptamer length, or structure. This design strategy could broadly extend the power, accessibility, and utility of numerous SSA-based biosensors.

Speeding up in Vitro Discovery of Structure-Switching Aptamers via Magnetic Cross-Linking Precipitation.

We report here a modified aptamer selection method, magnetic cross-linking precipitation (MCP)-SELEX, for highly efficient library enrichment and aptamer isolation. MCP-SELEX isolates bound aptamers via highly efficient chemical cross-linking between amino groups of target proteins and activated carboxylic acid groups on magnetic beads (>90% coupling efficiency). Importantly, MCP-SELEX avoids surface interferences in conventional target-fixed methods and substantially minimizes nonspecific binding. The enrichment efficiencies of MCP-SELEX for various proteins (PD-L1, ubiquitin, thrombin, and HSA) were all greatly higher than those of the conventional target-bound magnetic bead based-SELEX (MB-SELEX). Antithrombin aptamer with KD of 33 nM was successfully isolated by four rounds of MCP-SELEX. MCP-SELEX also enabled the efficient aptamer isolation by coupling with MB-SELEX or falling-off-SELEX. We identified structure-switching aptamers (SSAs) that specifically bind to HSA with low nanomolar dissociation constant via three rounds of MCP-SELEX and 1 round of falling-off-SELEX. Our HSA SSAs also have ∼3-fold higher specificity against streptavidin relative to thrombin SSAs discovered through falling-off-SELEX only. The enriched library has ∼78-fold higher signal-to-noise ratio (the number of DNAs eluted by 50 nM HSA divided by the number of DNAs self-dissociated in blank buffer) than that obtained by 4 rounds of direct falling-off-SELEX. We finally demonstrated the application of the selected SSA in fluorescent detection of HSA in urine with diagnostic required sensitivity and dynamic range. We expect that MCP-SELEX may be coupled with other selection methods to substantially accelerate aptamer discovery.

Long-Term Functional Stability of Functional Nucleic Acid-Gold Nanoparticle Conjugates with Different Secondary Structures.

Thiolated functional nucleic acid-gold nanoparticle conjugates (FNA-AuNPs) are the core recognition elements in biosensors. The long-term functional stability (LTFS) is critical for their practical applications and, however, has been overlooked. Here we report on the huge effects of multiple experimental factors on LTFS, including spacer- and buffer-composition, secondary structures of FNAs, and surface blocking. We quantitatively determined these effects by measuring the relative hybridization capacity (RHC, the relative amount of complementary DNA hybridized with the same amount of conjugates) for linear DNA-AuNP or the relative signal change generated by their function (RSC-F) for molecular beacon (MB) and G-quadruplex (G4)-AuNPs. There is a positive relationship between the spacer affinity [oligoadenine (A10) > oligothymine (T10) > oligoethlyene glycol (EG18)] of the linear DNA probes and the LTFS. The LTFS of linear DNA-AuNP in phosphate buffer (PB) was much better than that in Good's buffers such as HEPES, Tris, and MES. The secondary structure of FNAs also strongly impacted the LTFS, showing the substantially decreased LTFS from G4- to linear DNA- to MB-AuNPs, where EG18 spacer was used for all these conjugates. The surface blocking of FNA-AuNPs greatly improved the LTFS. We experimentally determined that the LTFS of FNA-AuNPs was directly related to the dissociation of DNAs caused by the in situ generated H2O2 due to the oxidase activity of AuNP and thereby oxidation of Au-thiol bonds. The oxidase activity of AuNP was favored at high temperature, low pH, high AuNP concentration, high Good's buffer concentration, and high salt concentration, corresponding well with the positive effects of high affinity spacer, PB, and surface blocking on the LTFS of FNA-AuNPs. Our study has implications on both fundamental surface science and practical applications.

Label-Free, Visual Detection of Small Molecules Using Highly Target-Responsive Multimodule Split Aptamer Constructs.

Colorimetric aptamer-based sensors offer a simple means of on-site or point-of-care analyte detection. However, these sensors are largely incapable of achieving naked-eye detection, because of the poor performance of the target-recognition and signal-reporting elements employed. To address this problem, we report a generalizable strategy for engineering novel multimodule split DNA constructs termed "CBSAzymes" that utilize a cooperative binding split aptamer (CBSA) as a highly target-responsive bioreceptor and a new, highly active split DNAzyme as an efficient signal reporter. CBSAzymes consist of two fragments that remain separate in the absence of target, but effectively assemble in the presence of the target to form a complex that catalyzes the oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid, developing a dark green color within 5 min. Such assay enables rapid, sensitive, and visual detection of small molecules, which has not been achieved with any previously reported split-aptamer-DNAzyme conjugates. In an initial demonstration, we generate a cocaine-binding CBSAzyme that enables naked-eye detection of cocaine at concentrations as low as 10 µM. Notably, CBSAzyme engineering is straightforward and generalizable. We demonstrate this by developing a methylenedioxypyrovalerone (MDPV)-binding CBSAzyme for visual detection of MDPV and 10 other synthetic cathinones at low micromolar concentrations, even in biological samples. Given that CBSAzyme-based assays are simple, label-free, rapid, robust, and instrument-free, we believe that such assays should be readily applicable for on-site visual detection of various important small molecules such as illicit drugs, medical biomarkers, and toxins in various sample matrices.

Amplification-by-Polymerization in Biosensing for Human Genomic DNA Detection.

A polymerization reaction was employed as a signal amplification method to realize direct visualization of gender-specific DNA extracted from human blood in a polymerase chain reaction (PCR)-free fashion. Clear distinction between X and Y chromosomes was observed by naked eyes for detector-free sensing purposes. The grown polymer films atop X and Y chromosomes were quantitatively measured by ellipsometry for thickness readings. Detection assays have been optimized for genomic DNA recognition to a maximum extent by varying the selection of the proper blocking reagents, the annealing temperature, and the annealing time. Traditional PCR and gel electrophoresis for amplicon identification were conducted in parallel for performance comparison. In the blind test for blood samples examined by the new approach, 25 out of 26 were correct and one was false negative, which was comparable to, if not better than, the PCR results. This is the first time our amplification-by-polymerization technique is being used for chromosome DNA analysis. The potential of adopting the described sensing technique without PCR was demonstrated, which could further promote the development of a portable, PCR-free DNA sensing device for point-of-need applications.

An efficient method to evaluate experimental factor influence on in vitro binding of aptamers.

Nucleic acid-based aptamers are promising alternative to antibodies, however, their selection process (SELEX) is challenging. A number of simulations and few experiments have been reported offering insights into experimental factors (EFs) that govern the effectiveness of the selection process. Though useful, these previous studied were either lack of experimental confirmation, or considered limited EFs. A more efficient experimental method is highly desired. In this study, we developed a fast method that is capable to quantitatively probe the influence of multiple EFs. Based on the fact that the aptamer enrichment efficiency is highly affected by background binding, the binding ratio between the numbers of specific aptamer binders and nonspecific (unselected library) binders per bead was used to quantitatively evaluate EF effects. Taking thrombin and streptavidin as models, three previously studied EFs (surface coverage, buffer composition, and DNA concentration) and four never-studied ones (surface chemistry, heat treatment, elution methodology and pool purity) were investigated. The EFs greatly affected binding ratio (ranging from 0.03 ±â€¯0.03 to 14.60 ±â€¯2.30). The results were in good agreement with the literature, suggesting the good feasibility of our method. Our study provides guidance for the choice of EFs not only for aptamer selection, but also for binding evaluation of aptamers.

Aptamer selection and application in multivalent binding-based electrical impedance detection of inactivated H1N1 virus.

The type A influenza viruses are the most virulent and variable human pathogens with epidemic or even pandemic threat. The development of sensitive, specific and safe field testing methods is in particular need and quite challenging. We report here the selection and practical utilization of the inactivated influenza virus-specific aptamers. The DNA aptamers against inactivated intact H1N1 virus particles were identified through the systematic evolution of ligands by exponential enrichment (SELEX) procedure. The discriminative aptamers and their truncated sequences showed selectively high affinity to inactive H1N1 virus and H3N2 virus with the Kd in the low nanomolar range and collective binding properties. The truncated sequences were first applied in a sandwich enzyme-linked oligonucleotide assay (ELONA) with a H1N1 detection limit (LOD, S/N = 3) of 0.3 ng/µL and then in an electrochemical impedance (EIS) aptasensor with more than 300 times improved LOD (0.9 pg/µL) and the excellent selectivity over other viruses (> 100 times). Therefore the developed aptasensors represent the safer, simpler, and possibly better virus-variation adaptable means of virus diagnostics.

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor.

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

Evanescent wave aptasensor for continuous and online aminoglycoside antibiotics detection based on target binding facilitated fluorescence quenching.

The biosensors capable for on-site continuous and online monitoring of pollutants in environment are highly desired due to their practical importance and convenience. The group specific detection of pollutants is especially attractive due to the diversity of environmental pollutants. Here we devise an evanescent wave aptasensor based on target binding facilitated fluorescence quenching (FQ-EWA) for the online continuous and group-specific detection of aminoglycoside antibiotics (AMGAs). In FQ-EWA, a fluorophore labeled DNA aptamer selected against kanamycin was used for both the target recognition in solution and signal transduction on optical fiber of EWA. The aptamers form multiple-strand complex (M-Apt) in the absence of AMGAs. The binding between AMGA and the aptamer disrupts M-Apt and leads to the formation of AMGA -aptamer complex (AMGA-Apt). The photo-induced electron transfer between the fluorophore and AMGA partially quenches the fluorescence of AMGA-Apt. The structure-selective absorption of AMGA-Apt over M-Apt on the graphene oxide further quenches the fluorescence of AMGA-Apt. Meanwhile, the unbound aptamers in solution assemble with the unlabeled aptamers immobilized on the fiber to form M-Apt. The amount of M-Apt on the fiber is inversely proportional to the concentration of AMGAs, enabling the signal-off detection of AMGAs from 200nM to 200µM with a detection limit of 26nM. The whole detection process is carried out in an online mode without any offline operation, providing a great benefit for system automation and miniaturization. FQ-EWA also shows great surface regeneration capability and enables the continuous detection more than 60 times.