Development of a sensitive and stable chemiluminescent immunoassay for detection of birch pollen allergic specific IgE based on recombinant Bet v1 protein.

Background Quantitative detection of allergens is of great significance for clarifying the cause, treatment, and prevention of allergy disease. Birch pollen is one of the most common inhalational allergens and Bet v1 is the major component allergen of birch allergen. This study aims to develop a stable and sensitive chemiluminescence immunoassay (CLIA) for the detection of birch pollen allergic specific IgE (sIgE) based on recombinant Bet v1 (rBet v1) protein. Methods rBet v1 protein was expressed in Escherichia coli and purified. Then rBet v1 was applied to detect sIgE in human serum. The performance of the established CLIA was evaluated and compared with Phadia rBet v1 fluorescence enzyme immunoassay (FEIA) system. Results The developed CLIA for sIgE to rBet v1 detection shows excellent performance. The assay showed a linear range from 0.1 to 100 IU/mL, with a low detection limit of 0.06 IU/mL. A total of 164 samples were evaluated by CLIA and compared with the results of FEIA. The positive, negative, and total coincidence rate was 90.6% (87/96), 91.2% (62/68), and 90.9% (149/164), respectively. The r-value of Spearman's rank correlation analysis was 0.935 (P < 0.001). The use of high levels of bilirubin (50 mg/dL), hemoglobin (400 mg/dL) and lipid (2000 mg/dL) didn't interfere with the results. Conclusions The proposed CLIA exhibits excellent performance for the detection of rBet v1 specific IgE. It can be a reliable tool for the early diagnosis of hypersensitivity.

An electrochemical aptasensor for ATP based on a configuration-switchable tetrahedral DNA nanostructure.

A novel electrochemical aptasensor for ATP was developed based on an aptamer-embedded configuration-switchable tetrahedral DNA nanostructure (TDN) and the formation of a G-quadruplex. This unique TDN was formed through the self-assembly of four specially designed single-stranded DNA (ssDNA) sequences (S1, S2, S3 and S4). The TDN was immobilized on the surface of a Au electrode through the thiol groups at the 5'-end of S1, S2 and S3. Five edges of the TDN were designed to form a double helix to preserve the structural robustness of the tetrahedron, while the ATP aptamer embedded sequence (S3) was designed to be located at the rest edge. The two terminals of S4 at the same edge were composed of two split G-quadruplex-forming sequences, which were non-complementary to the aptamer. This edge offered the configuration-switchable characteristic of the TDN. In the absence of ATP, the TDN remained in a relaxed state, and the G-quadruplex cannot form due to the large distance between the split G-quadruplex-forming sequences. However, in the presence of ATP, the aptamer combined with ATP and shortened the distance between the split sequences, resulting in the taut state of the TDN and the formation of a G-quadruplex at the edge. After the addition of hemin, the differential pulse voltammograms (DPVs) were used to quantify ATP. The sensor revealed a dynamic response range from 0.1 nM to 1 µM, with a detection limit of 50 pM. In addition, the specificity and practicability in real samples were also verified, indicating its potential applications.

A colorimetric ATP assay based on the use of a magnesium(II)-dependent DNAzyme.

A colorimetric assay for ATP is described that uses a strategy that combines the concept of split Mg(II)-dependent DNAzyme, split aptamer, and hybridization-induced aggregation of gold nanoparticles (AuNPs). Both ATP aptamer and Mg(II)-dependent DNAzyme are split into two fragments which are allocated to two well-designed DNA probes. The probes also possess mutually complementary stem sequences and spacer sequences. In the presence of ATP, the separated DNAzyme sequences in the two probes assemble via the synchronous recognition of ATP with two fragments of the aptamer. Then, the activated DNAzyme catalyzes multiple cycles of the cleavage of its substrate DNA sequence. The latter acts as a linker and induces the aggregation of two types of ssDNA-modified AuNP through the hybridization between the complementary sequences. Thus, the color of the AuNP solution remains red. However, in the absence of ATP, the detached aptamer cannot induce the assembly of DNAzyme to cleave the linker DNA. This results in the aggregation of AuNP and a concomitant color transition from red to purple. This ATP assay, performed at a wavelength of 530 nm, has a linear detection range that extends from 10 pM to 100 nM, with a detection limit of 5.3 pM. It was applied to the detection of ATP in human serum. Conceivably, the strategy has a wide scope in that it may be applied to the colorimetric detection of various other analytes through the split aptamer configuration. Graphical abstract Schematic presentation of colorimetric assay for adenosine triphosphate (ATP) based on the use of a split Mg(II)-dependent DNAzyme, a split aptamer, and by exploiting the hybridization-induced aggregation of gold nanoparticles that leads to a color change from red to purple.

An aptamer and functionalized nanoparticle-based strip biosensor for on-site detection of kanamycin in food samples.

A lateral flow strip biosensor for fast, sensitive, low-cost and on-site detection of kanamycin was developed by using kanamycin-specific aptamer-modified gold nanoparticles (AuNPs-apt) as a probe and oligonucleotide DNA1-modified silver nanoparticles (AgNPs-DNA1) as a signal amplification element. Through the complementary sequences of DNA1 and the aptamer, the AgNP-DNA1-apt-AuNPs complex can be formed and further captured on the test zone of the strip, where a capture probe DNA2 complementary to the 3'-terminal of DNA1 was immobilized. In the presence of kanamycin, it can competitively bind to the aptamer, and then inhibit the formation of the complex and the accumulation of AuNPs on the test zone. AuNPs-apt can finally be captured on the control zone via the specific binding between biotin and streptavidin. The assay avoids multiple incubation and washing steps and can be completed within 10 min. By observing the color change of the test zone, a qualitative detection for kanamycin can be achieved by the naked eye, with the visual limit of 35 nM. Meanwhile, a linear detection range of 1-30 nM with a low detection limit of 0.0778 nM for quantitative analysis can be achieved by using a scanning reader. The lateral flow strip biosensor exhibited high specificity and stability. Moreover, it was applied to detect kanamycin in various food samples, indicating its great potential in field testing.