Does size matter? Study of performance of pseudo-ELISAs based on molecularly imprinted polymer nanoparticles prepared for analytes of different sizes

The aim of this work is to evaluate whether the size of the analyte used as template for the synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) can affect their performance in pseudo-enzyme linked immunosorbent assays (pseudo-ELISAs). Successful demonstration of a nanoMIPs-based pseudo-ELISA for vancomycin (1449.3 g mol) was demonstrated earlier. In the present investigation, the following analytes were selected: horseradish peroxidase (HRP, 44 kDa), cytochrome C (Cyt C, 12 kDa) biotin (244.31 g mol) and melamine (126.12 g mol). NanoMIPs with a similar composition for all analytes were synthesised by persulfate-initiated polymerisation in water. In addition, core-shell nanoMIPs coated with polyethylene glycol (PEG) and imprinted for melamine were produced in organics and tested. The polymerisation of the nanoparticles was done using a solid-phase approach with the correspondent template immobilised on glass beads. The performance of the nanoMIPs used as replacement for antibodies in direct pseudo-ELISA (for the enzymes) and competitive pseudo-ELISA for the smaller analytes was investigated. For the competitive mode we rely on competition for the binding to the nanoparticles between free analyte and corresponding analyte-HRP conjugate. The results revealed that the best performances were obtained for nanoMIPs synthesised in aqueous media for the larger analytes. In addition, this approach was successful for biotin but completely failed for the smallest template melamine. This problem was solved using nanoMIP prepared by UV polymerisation in an organic media with a PEG shell. This study demonstrates that the preparation of nanoMIP by solid-phase approach can produce material with high affinity and potential to replace antibodies in ELISA tests for both large and small analytes. This makes this technology versatile and applicable to practically any target analyte and diagnostic field

Сенсорная система для определения сульфаметоксазола на основе молекулярно импринтированных полимерных мембран

Проблематика. Розробка сенсорних систем на основі штучних аналогів біологічних макромолекул є актуальною для сучасної аналітичної біотехнології, оскільки забезпечує нові ефективні експрес-методи детекції малих органічних молекул, в тому числі фармацевтичних препаратів. Мета дослідження. У роботі пропонується аналітична система для високоселективного та чутливого визначення сульфаметоксазолу на основі молекулярно імпринтованих полімерних (МІП) мембран, синтезованих із застосуванням методу полімеризації in situ у комбінації з методом комп’ютерного моделювання. Методика реалізації. Молекули сульфаметоксазолу, селективно адсорбовані штучними рецепторними сайтами у структурі МІП мембран, візуалізували завдяки їх здатності формувати забарвлені у коричневий колір комплекси після реакції з фериціанідом калію та нітропрусидом натрію в лужному середовищі. Результати дослідження. Межа визначення сульфаметоксазолу становила 2 мМ, а лінійний динамічний діапазон роботи сенсорної системи – 2–15 мМ, що дає змогу визначати сульфаметоксазол у фармацевтичних препаратах. Стабільність розроблених сенсорних систем на основі МІП становила принаймні 6 місяців, що значно перевищує стабільність аналогічних пристроїв на основі природних рецепторів. Висновки. Доведено придатність розроблених сенсорних систем для аналізу сульфаметоксазолу як у модельних, так і в реальних зразках (комерційно доступних фармацевтичних препаратах). Розроблені сенсорні системи характеризуються високою селективністю, чутливістю, портативністю та невисокою вартістю.Background. Development of sensor systems based on synthetic mimics of biological molecules will provide new effective express-methods for detection of small organic molecules, including pharmaceuticals, for modern analytical biotechnology. Objective. An analytical system for highly selective and sensitive detection of sulfamethoxazole based on molecularly imprinted polymer (MIP) membranes is proposed, synthesized using the method of in situ polymerization in a combination with the method of computational modeling. Methods. Sulfamethoxazole molecules, that were selectively adsorbed by the synthetic binding sites in MIP membranes structure, were visualized due to their ability to form brown-colored complexes after reaction with potassium ferricyanide and sodium nitroprusside in alkaline media. Results. The limit for sulfamethoxazole detection comprised 2 mM, while the linear dynamic range – 2–15 mM, which allows one to detect sulfamethoxazole in pharmaceutical preparations. Stability of the developed MIP-based sensor systems was estimated as at least 6 months, which significantly increases stability of analogous devices based on natural receptors. Conclusions. Applicability of the developed sensor systems for the analysis of sulfamethoxazole in both model solutions and real samples (commercial pharmaceutical preparations) was proven. The developed systems are characterized with high selectivity, sensitivity, small size and low cost.Проблематика. Разработка сенсорных систем на основе искусственных аналогов биологических макромолекул является актуальной для современной аналитической биотехнологии, поскольку обеспечивает новые эффективные экспресс-методы детекции малых органических молекул, в том числе фармацевтических препаратов. Цель исследования. В работе предлагается аналитическая система для высокоселективного и чувствительного определения сульфаметоксазола на основе молекулярно импринтированных полимерных (МИП) мембран, синтезированных с использованием метода полимеризации in situ в комбинации с методом компьютерного моделирования. Методика реализации. Молекулы сульфаметоксазола, селективно адсорбированные синтетическими рецепторными сайтами в структуре МИП мембран, визуализировали благодаря их способности формировать окрашенные в коричневый цвет комплексы после реакции с феррицианидом калия и нитропруссидом натрия в щелочной среде. Результаты исследования. Предел обнаружения сульфаметоксазола составил 2 мМ, а линейный динамический диапазон сенсорной системы – 2–15 мМ, что дает возможность определять сульфаметоксазол в фармацевтических препаратах. Стабильность разработанных сенсорных систем на основе МИП составила по крайней мере 6 месяцев, что значительно превосходит стабильность аналогичных приборов на основе природных рецепторов. Выводы. Доказана возможность использования разработанных сенсорных систем для анализа сульфаметоксазола как в модельных, так и в реальных образцах (коммерчески доступных фармацевтических препаратах). Разработанные сенсорные системы характеризуются высокой селективностью, чувствительностью, портативностью и невысокой стоимостью

Interfacial Molecular Imprinting in Nanoparticle-Stabilized Emulsions

A new interfacial nano and molecular imprinting approach is developed to prepare spherical molecularly imprinted polymers with well-controlled hierarchical structures. This method is based on Pickering emulsion polymerization using template-modified colloidal particles. The interfacial imprinting is carried out in particle-stabilized oil-in-water emulsions, where the molecular template is presented on the surface of silica nanoparticles during the polymerization of the monomer phase. After polymerization, the template-modified silica nanoparticles are removed from the new spherical particles to leave tiny indentations decorated with molecularly imprinted sites. The imprinted microspheres prepared using the new interfacial nano and molecular imprinting have very interesting features: a well-controlled hierarchical structure composed of large pores decorated with easily accessible molecular binding sites, group selectivity toward a series of chemicals having a common structural moiety (epitopes), and a hydrophilic surface that enables the MIPs to be used under aqueous conditions

Biotin–specific synthetic receptors prepared using molecular imprinti

The composition of new molecularly imprinted polymers (MIPs) specific for biotin was optimised using molecular modelling software. Three functional monomers: methacrylic acid (MAA), 2-(trifluoromethyl)acrylic acid (TFAA) and 2-acrylamido- 2-methyl-propanesulfonic acid (AMPSA), which demonstrated the highest binding scores with biotin, were tested on their ability to generate specific binding sites. The imprinted polymers were photografted to the surface of polystyrene microspheres in water. The affinity of the synthetic "receptor" sites was evaluated in binding experiments using horseradish peroxidase-labelled biotin. A good correlation was found between the modelling results and the performance of the materials in the template rebinding study. The dissociation constants for all MIPs were 1.4-16.8 nM, which is sufficient for most analytical applications where biotin is used as a label

Solid phase extraction of alpha-tocopherol and other physiologically active components from sunflower oil using rationally designed polymers

A rationally designed polymer (RDP) capable of recognizing α-tocopherol and other minor components in sunflower oil has been produced. It is known that sunflower oil is a source of various physiologically active compounds. Unfortunately, they are present in very minor quantities which make their purification from the complex oil matrix problematic. An extraction method presented here was developed with particular attention to the selectivity, efficiency and precision of the extraction process. The methacrylic acid-based RDP in combination with the optimised purification method allowed the extraction of α-tocopherol with 94% recovery. The synthesised polymer was used successfully to extract α-tocopherol together with other essential minor components of sunflower oil without any pre-treatment step. According to GC/MS, the compounds ‘harvested’ from sunflower oil using the developed polymer included palmitic, oleic and linoleic acids, α-tocopherol, campesterol, stigmasterol, and β-sitosterol

Ice matrix in reconfigurable microfluidic systems

Microfluidic devices find many applications in biotechnologies. Here, we introduce a flexible and biocompatible microfluidic ice-based platform with tunable parameters and configuration of microfluidic patterns that can be changed multiple times during experiments. Freezing and melting of cavities, channels and complex relief structures created and maintained in the bulk of ice by continuous scanning of an infrared laser beam are used as a valve action in microfluidic systems. We demonstrate that pre-concentration of samples and transport of ions and dyes through the open channels created can be achieved in ice microfluidic patterns by IR laser-assisted zone melting. The proposed approach can be useful for performing separation and sensing processes in flexible reconfigurable microfluidic devices

Attenuation of Vibrio fischeri Quorum Sensing Using Rationally Designed Polymers

A first attempt to attenuate the quorum sensing (QS) of a marine heterotroph microorganism, Vibrio fischeri, using signal molecule-sequestering polymers (SSPs) is presented. A set of rationally designed polymers with affinity toward a signal molecule of V. fischeri, N-(?-ketocaproyl)-l-homoserine lactone (3-oxo-C6-AHL) was produced. It is reported that computationally designed polymers could sequester a signal molecule of V. fischeri and prevent QS-controlled phenotypes (in this case, bioluminescence) from being up-regulated. It was proven that the attenuation of bioluminescence of V. fischeri was due to sequestration of the signal molecule by specific polymers and not due to the toxicity of polymer or nonspecific depletion of nutrients. The ability to disrupt the bacterial communication using easy to synthesize and chemically inert polymers could provide a new concept for the development of pharmaceuticals and susceptible device coatings such as catheters