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.

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.

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.

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.

The γ-gliadin-like γ-prolamin genes in the tribe Triticeae.

The γ-prolamins are important components of seed storage proteins in wheat and other Triticeae species. Here, the γ-prolamin genes from the diploid Triticeae species were systemically characterized. Most of the γ-prolamins (except 75 K γ-secalins) characterized were defined as γ-gliadin-like γ-prolamins, since they shared same characteristic model structure with γ-gliadins. Over one-third of these putatively functional γ-prolamin peptides contained different number of cysteine residues as compared to the eight residues present in γ-gliadins. Sequence polymorphism and linkage disequilibrium analyses showed the conservation of γ-prolamin genes in Triticeae species under evolutionary selection. Phylogenetic analyses indicated that these γ-prolamin genes can not be clearly separated according to their genomic origins, reflecting the conservation of γ-gliadinlike γ-prolamin genes after the divergence of Triticeae species. A screening of coeliac disease (CD) toxic epitopes shows that the γ-prolamins from some other genomes contain much fewer epitopes than those from the A, S (B) and D genomes of wheat. These findings contribute to better understanding of γ-prolamin family in Triticeae and build a ground for breeding less CD-toxic wheat cultivars.