ORIGINAL PAPER A novel separation and enrichment method of 17b-estradiol using aptamer-anchored microbeads Gioi Dong Huy • Na Jin • Bin-Cheng Yin • Bang-Ce Ye Received: 6 May 2010 / Accepted: 30 July 2010 / Published online: 24 August 2010 Ó Springer-Verlag 2010 Abstract The estrogenic compound 17b-estradiol (E2) is widely studied for its potential endocrine disruption effects. Due to the low level of E2 present in the environment, it is highly desirable to develop a sensitive and efficient sepa- ration and enrichment method for E2 analysis. In this paper, we proposed a novel E2 preconcentration method using anti-E2 aptamer-anchored isothiocyanate-modified beads (NCS beads). The glass beads are chemically mod- ified with primary amino group, and then treated with phenylene diisothiocyanate (PDITC) to generate an iso- thiocyanate group, which is reactive towards the amine group. The amino-modified anti-E2 aptamer can be easily covalently immobilized onto the as-prepared NCS beads. The experimental results demonstrated that the aptamer affinity microbeads could selectively retain and separate E2 compound. The effects of the operation parameters on retention of E2, including washing condition, eluting con- dition, the number of beads, and the incubation time were investigated. Moreover, high-performance liquid chroma- tography with preconcentration of E2 on the aptamer affinity microbeads was applied to detect the E2 in the spiked water samples and obtained a good recovery. Keywords 17b-Estradiol  Separation  Enrichment  Affinity chromatography  Aptamer Introduction It is well known that the endocrine disrupting compounds (EDCs) have harmful effects on aquatic organisms [1–4]. They are closely related to the normal endocrine function of animals and humans [5]. 17b-estradiol (E2), the most active estrogen, has been widely used in animal fattening for its anabolic effects [6]. The chronic exposure of humans to E2 will cause drastic problems through food chain [7]. Considerable interests are focused on the detection of E2, such as instrumental analysis methods [8, 9] and biosensor methods [10, 11]. High-performance liquid chromatography with UV absorbance detection is frequently used to detect 17b-estradiol in complex envi- ronmental samples, but to some extent, its lack of selec- tivity necessitates great care in sample preparation and enrichment to avoid interferences. The most commonly method for the E2 preconcentration is solid phase extraction (SPE), which is routinely used for preconcen- tration and clean up in the analysis of biological and environmental samples. The classical SPE sorbents (C8, C18, etc.) retain analytes by non-selective hydrophobic reaction, which leads to a partial coextraction of inter- fering substances, and further purification procedure is sometimes still required to remove coextractant. A highly selective stationary phase may eliminate these problems and facilitate development of a simple efficient enrich- ment method for rapid selection and purification of compounds from complex mixtures, such as affinity SPE. The antibody-based SPE formats are perhaps the most common because of their high selectivity, high affinity, and the variety of antigens that can be targeted. However, their applications in real samples are limited due to the instability of natural antibodies. Therefore, it is highly desirable to develop a rapid, sensitive and cost-effective G. Dong Huy  N. Jin  B.-C. Yin  B.-C. Ye (&) Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Meilong Road 130, Shanghai 200237, China e-mail: bcye@ecust.edu.cn 123 Bioprocess Biosyst Eng (2011) 34:189–195 DOI 10.1007/s00449-010-0460-4