Reserach Express@NCKU - Articles Digest

Estrogen (17β-estradiol, E2) is an ovarian hormone necessary for the development of secondary sexual characteristics and function of the reproductive system in females. The estrogen action, however, is complicated by the interaction of E2 with two receptors, ERα and ERβ. The binding of E2-activated ERs to estrogen responsive element (ERE) in the promoter regions of target genes induces gene expression and cell proliferation but inhibits cell apoptosis, which then alter cellular functions. In clinical practice, antiestrogens have been used to treat breast cancer. Estrogen supplement is also used for postmenopausal women. To eliminate the complications associated with these medicines, it is desirable to develop more specific antiestrogens for patients with related diseases. The action mechanisms of E2 are associated with at least two kinds of specific interaction: small molecules E2 with ER protein, and ER protein with ERE DNA molecules. Conventionally, these two bindings are analyzed separately based on different platforms: Radio-isotope labeled [3H]-estrogen is used to estimate the ligand binding and electrophoretic mobility shift assay is used to investigate ERE binding. These methods, however, suffer from one or more disadvantages such as the use of hazard radio isotopes, slow process, intensive labor handling, large sample quantity and difficulties for quantification. Therefore, alternative analytical platforms are highly demanded.

We would like to take advantages of the microfluidics platforms in less sample/reagent consumptions and rapid analysis to develop enabling assays and one of the particular attentions has been paid to microchip electrophoresis. In addition to separation, a new focus would be for characterizing chemical equilibria or affinity interactions. Using this approach, the two-step binding mechanism of E2 and ERE can be investigated in free solutions under non-denaturing conditions. Like many other chip-based assays, however, increasing detection sensitivity and reducing irreversible adsorption on chip surface are challenging tasks since the concentration of biological samples are low. In this study, a novel on-chip concentration and surface coating methods are developed to analyze E2 and ERE binding on glass substrates independently.

Figure 1

To perform electrophoretic separation on a microchannel, the volume of sample that can be introduced into a microchannel for analysis is limited to sub nL to prevent zone diffusion that will degrade the separation efficiency. This characteristic grants the chip-based separation method a superior advantage in low sample volume required for analysis but also greatly limits the detection sensitivity for samples with low concentration. We combined a semi-hydrodynamic (SHD) injection with a high salt sample stacking/sweeping method to introduce a large sample plug (50 nL) into a microchannel and then compressed it into a narrow band (near sub nL) before sample separation and detection. We used a simple cross microchannel that is 2 cm and 6 cm in length; the width and depth of all microchannels are 100 and 20μm, respectively. As shown in Figure 1, this method uses a 100 L syringe pump for introducing a high-salt sample (100 mM KCl) plug into a cross channel while the sample is being focused by application of voltage to the two side arms. Moreover, as depicted in Figure 2, a higher concentration of micelles (sodium cholate) is created in the zone boundary by field stacking; sweeping occurs when micelles in the running buffer penetrate the sample zone devoid of the micelle, and the micelles collect the hydrophobic sample such as E2 into a narrow concentrated zone. Subsequently, the free E2 and E2/ER complex are separated and detected as the band migrates along the separation microchannel and the relative binding strength can be evaluated based on the relative intensitivity of the free and complex band (Figure 3). The detection sensitivity of ligand-receptor binding was increased by 2-3 orders of magnitudes using this method. The buffer system also acts to solubilize the compounds and to separate the free and complex estrogens. The ligand binding force was quantified based on the attenuation of the complex signal.

Figure 2

For ERE binding, wall adsorption effect is severe and we assemble polyethylene glycol (PEG)-coated glass chips using layer by layer method to sustain the analyte in the free solution. The PEG-coated glass chip bears neutral surfaces against the adsorption of acidic DNA molecules and basic estrogen receptor (ER) proteins, and therefore, irreversible surface adsorption was efficiently minimized. As depicted in Figure 4, the glass microchip was first coated with 3-aminopropyltriethoxysilane (APTES) by alcohol condensation and then topped with PEG by EDC/NHS chemistry. Combined with SHD injection method, the ER-ERE binding force was reflected in the shift of the migration time for FITC-labeled ERE molecules in a sieving matrix. As listed in Table 1, using this approach, the dissociation constant for both recombinant ER α and ER β with the consensus ERE (cERE) sequence (5’-GGTCAGAGTGACC-3’) was determined to be around 6 and 12 nM, respectively. Moreover, the estrogenic compound-mediated dissociation constants of a ERE 1576 (5’-GACCGGTCAGCGGACTCAC-3’) sequence deduced from vascular endothelial growth factor (VEGF) gene with the extracted ER from treated and untreated A549 bronchioloalveolar carcinoma cells were also determined. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17-Estradiol (1.70 nM), Diethylstilbestrol (0.14 nM), and Genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.

Such chip-based assays bear many advantages such as minimum reagent required, speedy analysis and automation. This method should hold great promises for dug screening or for the characterization of lead compounds, particularly for those with limited amount.

Figure 3

Figure 4

Table 1 Agonist/antagonist mediated dissociation constants determined by microchip electrophoresis