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An aptamer-amphiphile was designed that binds to β-lactoglobulin (β-LG), a major allergen from cow’s milk. For this work, a 23-nucleotide ssDNA aptamer β-LG-23, capable of forming antiparallel G-quadruplexes was used, and its specificity and binding affinity of 22 ± 2 nM for β-LG were evaluated via enzyme-linked apta-sorbent assay (ELASA). The β-LG-23 aptamer was synthesized as an amphiphile by conjugating it to a C16double tail via different spacers, and the effect of the spacers on the binding affinity and secondary structure of the aptamer was investigated. From all amphiphiles tested, direct conjugation of the aptamer to the tail gave the lowest binding affinity to β-LG (37 ± 2 nM), while maintaining the antiparallel G-quadruplex secondary structure of the aptamer. As a proof of concept, the β-LG-23 aptamer-amphiphile was used to decorate the interface of a liquid crystal (LC) and effectively detected 10 nM or 0.18 ppm of β-LG with a 20 min equilibration time, thus demonstrating that it has the potential to be used for fast and label-free detection of β-LG.

ABSTRACTIntroduction: The physiologic importance of fast CO2/HCO3−interconversion in various tissues requires the presence of carbonic anhydrase (CA, EC 4.2.1.1). Fourteen CA isozymes are present in humans, all of them being used as biomarkers.Area covered: A great number of patents and articles were focused on the use of CA isozymes as biomarkers for various diseases and syndromes in the recent years, in an ascending trend over the last decade. The review highlights the most important studies related with each isozyme and covers the most recent patent literature.Expert opinion: The CAs biomarker research area expanded significantly in recent years, shifting from the predominant use of CA IX and CA XII in cancer diagnostic, staging, and prognosis towards a wider use of CA isozymes as disease biomarkers. CA isozymes are currently used either alone, in tandem with other CA isozymes and/or in combination with other proteins for the detection, staging, and prognosis of a huge repertoire of human dysfunctions and diseases, ranging from mild transformation of the normal tissues to extreme shifts in tissue organization and function. The techniques used for their detection/quantitation and the state-of-the-art in each clinical application are presented through relevant clinical examples and corresponding statistical data.

Hypoxia is a common feature of solid tumors contributing to resistance to chemotherapy. Selective delivery of chemotherapeutic drugs to hypoxic tumor niche remains an unsolved issue. For this purpose, we constructed a gold nanoplatform targeting carbonic anhydrase IX (CA IX) epitope, which is overexpressed in hypoxic tumor cells versus in normal tissues. We designed compatible low-molecular weight carbonic anhydrase inhibitor (CAI) ligands and doxorubicin (Dox) ligands and optimized protocols for efficient decoration of gold nanoparticles (Au NPs) to achieve both good targeting ligand density and optimum drug loading, while preserving colloidal stability. The optimized Dox-HZN-DTDP@Au NPs-LA-PEG2000-CAI (THZN) nanoplatform was proved to be very efficient toward killing HT-29 tumor cells, especially under hypoxic conditions, as compared with the nontargeting nanoplatform. This also mediated the effective release of doxorubicin in the lysosomes following internalization, as revealed by confocal microscopy. Furthermore, using tumor spheroids as a representative model for hypoxic solid tumors, our THZN nanoplatform enhanced the selective delivery of doxorubicin up to 2.5 times and minimized chemoresistance, showing better tumor drug penetration as compared to that in free drug treatment. Our technology is the first CA IX-targeting gold nanoplatform for efficient delivery of doxorubicin to hypoxic tumors in a controlled fashion, with the perspective to improve the therapy of solid tumors and minimize chemoresistance.