Novel Biological Samples Pretreatment Based On Restricted Access Magnetic Microspheres

With growing demand for fast analysis in the fields of pharmaceutical industry and biomedical research, the pretreatment of biological samples is of great importance in establishing a reliable and accurate analytical method. Magnetic solid phase extraction (MSPE) with the advantages of fast and simple procedures and being amenable to high-throughput operation has gradually taken the place of traditional technologies including liquid-liquid extraction (LLE) and solid-phase extraction (SPE). However, the fouling of proteins on the surface of magnetic particles gives rise to particles aggregation, leading to declined binding capacity for analytes and reusability of the particles.Recently, restricted access SPE has been developed to be as a type of on-line extraction technology enabling the direct enrichment of analytes while eliminating macromolecules from biological fluids. One of major advantages over traditional SPE is that the external surface of restricted access meterials (RAMs) has hydrophilic groups combined with limited pore size as barriers which prevent surface adsorption and pore channel blocking. The interior surface modified with hydrophobic and ion-exchange groups can directly extract low weight targets from biological samples. Therefore, the RAM has been widely applied for pretreatment technology in the fields of biological and environmental analysis. However, the only single biological sample can be treated from enrichment to analysis under this kind of on-line extraction mode, which will cause the low availability of the instrument and cannot satisfy the requirement of high-throughput in biological fluids.In order to meet the need of high-throughput of biological samples and to provide solutions for the particle fouling due to protein adsorption on the surface, efforts have been made in this thesis to develop preparation methods by which the restricted access properties are incorporated into the magnetic separation materials so as to provide novel restricted access materials for magnetic solid-phase extraction.The major results presented in this thesis are outlined as follows:1. The magnetic silica particles served as scaffolds were first modified with diol groups, which were then converted to octadecyl esters through reaction with stearoyl chloride. In the second step, the octadecyl esters on the exterior surface were hydrolyzed by the action of lipase to yield magnetic RAM particles with hydrophobic reverse-phase ligands on the inner surface and biocompatible diol groups on the outer surface. The restricted access behavior of the resulting materials was confirmed by differential binding of small molecules such as methotrexate (MTX), calcium folinate (CF), and folic acid (FA) relative to bovine serum albumin (BSA). While the three model drugs were bound to the magnetic particles with high affinity, the adsorption of the proteins was markedly reduced due to size exclusion effect. The utility of the magnetic particles for sample preparation was tested in solid-phase extraction of MTX, CF and FA from spiked human serum and the effects of the SPE conditions on the recovery of the analytes were systematically studied. Moreover, the magnetic particle-based sample preparation procedures coupled with reversed-phase liquid chromatography analysis were developed and validated. This dual functionality material may be adapted in automated and high-throughput protocols for routine analysis of a large volume of clinical samples.2. Owing to the weak binding strength between three model drugs and the materials from above method, porous magnetic silica was first modified with epoxy groups, which were reacted with polymeric acid with particle size 50μm to yield only diol groups at the outer surface. In the second step, three compounds, octadecylamine, sodiumhydrogensulfite and triethylamine hydrochloride, respectively, were reacted with epoxy groups at the inner surface to give rise to magnetic RAM particles with octadecyl, sulfonic and quaternary ammonium on the inner surface and diol groups on the outer surface. This synthesis method can avoid the single type with only modified into alkyl groups on the inner surface aboved as lipase hydrolysis. The model molecules above mentioned were still used to compare the binding capacity of three magnetic RAMs through Langmuir adsorption isotherm curves. The results showed that the adsorption amount of materials with sulfonic and quaternary ammonium on the inner surface was significantly higher than that with octadecyl groups, implying that MTX and its structural analogs can be extracted more efficiently by ion exchange model. Furthermore, the open-ring methods based on polymeric acid has more advantageous in the interior versatilities over that based on lipase hydrolysis.3. In order to enhance adsorptive capacity on biological polypeptides with high molecule weight, the salt impregnation was carried out and followed by calcination at different temperatures to produce wide poreγ-Fe₂O₃@SiO2 magnetic silica. The optimal wide pore conditions were defined at the temperature of 300℃and 400℃ through the characterization of morphology, crystallization, magnetism and surface area of magnetic silica particles. The wide-pore material was then further modified into RAM particles with octadecyl groups on the inner surface and diol groups on the outer surface by the open-ring reaction of polymeric acid. Three kinds of different molecule weight of biological polypeptides, including Thymopentin (TP-5),Insulin and BSA, were chosen as model compounds to perform the adsorption, which indicated that the wide pore materials were higher binding capacity on Insulin and BSA, and the ability of exclusion with different degree. This also offers the potential high selectivity for extraction and enrichment of different molecule weight of functional targets from complex biological fluids.4. In order to develop novel methods to prepare magnetic RAM, porousγ-Fe₂O₃ microspheres were chosen as magnetic core, cetyltrimethylammonium bromide (CTAB) as structure direction agent, and layer by layer coating method based on sol-gel principles was adopted to modify through different types of silanes with functional groups, which yielded core-shell magnetic RAM particles with amino, sulfonic, phenylsulfonic or caroxyl groups at the inner surface and diol groups at the outer surface. First, the magnetic RAMs with amino groups at the inner surface was used as model material, all kinds of characterization tools including FT-IR, HRTEM, and fluorescence quenching and magnetism curves had been used to confirm that the template was removed effectively to form surface shell and porous channel together with restricted access properties. The adsorption experiments of macromolecules BSA and basic model small weight compounds such as Terbutaline (TER), Salbutamol (SAL) and Clenbuterol (CLB) were chosen to exhibit the dual surface functional properties from the latter three core shell magnetic RAMs based on ion-exchange principles. The results indicated that the one kind, or over two kinds and even the whole silanes agent could be directly grafted onto the inner surface of particles, which have more selective and versatile than above methods. This also provides a novel thought for preparation of magnetic RAMs.