Assessment of tumor metastasis by the direct determination of cell-membrane sialic acid expression.

Sialic acid (SA) is an anionic monosaccharide that frequently occurs at the termini of glycan chains and provides many opportunities for the assessment of both normal and pathological cell processes. It is generally present in tumorassociated carbohydrate antigens, including those clinically approved as tumor markers. Accordingly, the overexpression of SA on cell membranes has been implicated in the malignant and metastatic phenotypes of various types of cancer. Therefore, SA is an important molecular target for diagnostic and therapeutic approaches. The installation of SA-specific ligands enables reagents to target highly sialylated or tumor cells. Alternatively, monitoring of the cellsurface expression of SA should provide rational indexes of dynamic changes in pathological conditions and other SAassociated biological events. We previously developed a method for the potentiometric detection of SA by exploiting the reversible and specific nature of the binding between phenylboronic acid (PBA) and SA. A gold electrode modified with PBA and with a carefully optimized dissociation constant (or pKa value) was able to quantify SA present in the free state as well as cell-surface SA under physiological aqueous conditions. The observed ability of the electrode to differentiate altered levels of SA expression on the surface of rabbit erythrocytes is relevant to the diagnosis of insulin-dependent diabetes mellitus (IDDM). The approach provided a new rationale for the label-free, noninvasive (enzyme-free and operative on living cells), and real-time determination of SA. Herein we show that the technique can also be applied to the analysis of tumor malignancy and the degree of metastasis. PBA derivatives are able to form reversible cyclic boronates with 1,2-diols, 1,3-diols, and polyols: hallmark structures of the majority of glycans. Because of this property, PBA has quite a history as a synthetic ligand for these molecules. It is usually observed that these complexes have a stabilizing effect only if PBA is disassociated (at pH values above the pKa value), [6] whereas those formed between nondissociated PBA and sugars are unstable with high susceptibility to hydrolysis. However, as an exception, a complex formed between nondissociated PBA and SA is remarkably stable owing to its special binding modalities, some aspects of which have been clarified previously. As a result, a PBA with an appropriate pKa value can provide a molecular basis for selective recognition of SA among other saccharides under physiological conditions (see the Supporting Information). A procedure for the surface modification of a gold electrode with PBA was described previously. Briefly, a self-assembled monolayer (SAM) of 10-carboxy-1-decanethiol was first formed on a gold electrode. Next, a reaction between the terminal carboxyl groups and 3-aminophenylboronic acid resulted in the introduction of meta-amidesubstituted PBA on the SAM surface. Both quartz crystal microbalance (QCM) and ellipsometric measurements confirmed stoichiometric monolayer formation at each step of the reaction (see the Supporting Information). The surface PBA moiety had an apparent pKa value of about 9.5, as judged from pH-dependent changes in its threshold voltage (VT; see the Supporting Information). We could therefore safely conclude that it was not dissociated at the physiological pH value (7.4) and would be SA-specific under such conditions. The electrode was then linked to a field-effect-transistor (FET) gate for the real-time monitoring of charge-density changes on the electrode. In this configuration, a carboxyl anion of SA can be detected as a positive-direction shift of the VT value of the FET. Owing to the nature of the field effect, FET-based charge detection is possible only within a distance corresponding to the electrical double layer or the Debye length, which is no greater than a few nanometers even under conditions of minimized ionic strength. This requirement should be compatible with the purpose of detecting cellsurface SA moieties, which generally dominate the termini of the glycan chains, as described earlier. Besides, the tumoror metastasis-associated overexpression of SA is usually found in the form of polysialylation. Such a sequential arrangement of target SA units (as an SA homopolymer) on the glycanchain termini may help to enable the precise reflection of altered levels of SA expression. Moreover, the fact that the technique is limited to short detection distances could beneficially restrict charge detection to molecules that are truly (covalently) bound to the electrode surface within the vicinity of the Debye length (i.e., PBA-bound SA) and exclude other charges bound through nonspecific or noncovalent interactions. To demonstrate the ability of the electrode to assess malignancy or metastasis of a tissue specimen, metastatic murine melanoma cells expressing luciferase (B16-F10-Luc[*] Dr. A. Matsumoto, Dr. H. Cabral, N. Sato, Dr. K. Kataoka, Dr. Y. Miyahara Centre for NanoBio Integration, The University of Tokyo Hongo 7-3-1, Bunkyo-ku, Tokyo (Japan) Fax: (+ 81)29-860-4506 E-mail: miyahara.yuji@nims.go.jp

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