Akira Matsumoto

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

A totally synthetic glucose responsive gel operating in physiological aqueous conditions

Here described is a phenylboronic acid (PBA) based glucose-responsive hydrogel operating under physiological pH and temperature, a material potentially applicable to a totally synthetic smart insulin delivery system to treat diabetes.

STAT5 Activation Correlates with Erythropoietin Receptor-mediated Erythroid Differentiation of an Erythroleukemia Cell Line

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. EPO has been shown to activate the JAK2-STAT5 pathway in various hematopoietic cell lines, although the physiological role of this pathway is unclear. We have previously shown that epidermal growth factor activates a chimeric receptor bearing the extracellular domain of the epidermal growth factor receptor linked to the cytoplasmic domain of the EPO receptor, resulting in proliferation of interleukin-3-dependent hematopoietic cells and erythroid differentiation (globin synthesis) of EPO-responsive erythroleukemia cells. In the present study, we introduced various deletion and tyrosine to phenylalanine substitution in the cytoplasmic domain of the chimeric receptor and expressed these mutant chimeras in an EPO-responsive erythroleukemia cell line, ELM-I-1. Mutant chimeric receptors retaining either Tyr343 or Tyr401 could activate STAT5, judged by tyrosine-phosphorylation of STAT5 and induction of CIS, a target gene of STAT5. These mutants were able to induce erythroid differentiation. However, a chimeric receptor containing both Y343F and Y401F mutations could not activate STAT5 nor induce erythroid differentiation. Thus, Tyr343 or Tyr401 of the EPO receptor are independently necessary for erythroid differentiation as well as STAT5 activation. Moreover, exogenous expression of dominant-negative STAT5 suppressed EPO-dependent erythroid differentiation. These findings suggest that STAT5 plays an important role in erythroid differentiation through the EPO receptor cytoplasmic domain.

Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

Chemical-to-Electrical-Signal Transduction Synchronized with Smart Gel Volume Phase Transition.

A stimulus-responsive polymer gel designed on a field-effect transistor gate undergoes a reversible volume phase transition in response to a specific biomolecule. An abrupt permittivity change at the gel/gate interface during the transition gives rise to a chemical to electrical signal conversion; the signal is thus detectable via a transistor without the limit of the Debye length.

Electrical visualization of chemo-mechanical signal transduction using a smart gel-modified gate field effect transistor

A field effect transistor (FET) whose gate surface has been modified with a stimuli-responsive smart polymer gel can visualize the kinetics of the chemo-mechanical signal transduction as a mode of its altered electrical characteristics without any labels.

Microbial Genetic Analysis Based on Field Effect Transistors

In this chapter, potentiometric detection methods for microbial DNA involved recognition events by use of genetic field effect devices will be described. Fundamental principles of field effect devices and the technical background with their ongoing applications in the field of bio-sensor technologies, termed bio-FET, will be first introduced. Then concept of genetic field effect transistor will be described with emphasis on their fabrication, characteristics, and recent applications to microbial Single Nucleotide Polymorphysms (SNPs) Analysis as well as DNA sequencing. By comparing to other conventional methods, technical significance and future perspective of the genetic field effect transistor will also be discussed in detail.

Preliminary characterization of a glucose-sensitive hydrogel

We present proof-of-concept studies that display the potential for using a glucose-sensitive hydrogel as a continuous glucose sensor. A study to characterize the swelling ratio of the hydrogel at normal physiological and pathological hyperglycemic glucose levels was performed. The hydrogel exposed to the hyperglycemic glucose solution had a higher equilibrium swelling ratio than the hydrogel exposed to the normal glucose concentration solution. The diffusivity of a small molecule, fluorescein isothiocyanate (FITC), through a hydrogel exposed to a hyperglycemic solution was determined using fluorescence recovery after photobleaching (FRAP). The diffusivity was found to be 4.2 × 10−14 m2/s, a value approximately four orders of magnitude smaller than the diffusivity of FITC in glucose solution. The permeability of the hydrogel after equilibration in a hyperglycemic solution was found to be 5.1 × 10−17 m2, in the range of 2–4% agarose gels.

Detection of biomolecular recognition using Bio-transistors

We have been investigating direct interaction between biomolecular charges and charged carriers in semiconductor materials. Field effect transistors have been used to detect biomolecular recognition based on electrostatic interaction as shown in Fig. 1. In the case of genetic FET, oligonucleotide probes are immobilized on the surface of the gate insulator. The cycle of single-base extension and measurement of the VT was repeated iteratively to determine the base sequence of the target DNA. As a result, the positive VT shifts could be detected in accordance with the base sequence of the target DNA. We also propose an oocyte-based field effect transistor (oocyte-based FET) for drug transport analysis, in which target transporters are expressed at the cell membrane of the oocyte. Non-invasive monitoring of the uptake kinetics of substrates mediated by membrane-bound transporters can be realized with oocyte-based FET. Discrimination of transporting ability among genotypes of the transporters could be realized using the oocyte-based FET. We also develop a label free, potentiometric method to detect cell surface sialic acid (SA) using phenylboronic acid (PBA) compound integrated into the form of self-assembled monolayer (SAM) on a field effect transistor (FET) extended gold gate electrode. Conceptual scheme for label free detection of cell surface SA utilizing the reversible and covalent interaction with phenylboronic acid (PBA) compound is shown in Fig. 2. A self-assembled monolayer of 10-carboxy-1-decanethiol was first formed on a gold gate electrode followed by a condensation reaction with an amino group functionalized PBA (3-aminophenylboronic acid) in order to obtain a PBA-modified gold electrode. This was then lined to a field effect transistor (FET) gate for real-time monitoring of the charge density changes taking place on the electrode when binding with anionically charged SA. Due to predominant binding between undisassociated PBA and SA at pH 7.4, we found that carboxyl anions of SA were exclusively detectable among other glycan chain constituent monosaccharides, as the change in threshold voltage (VT) of the PBA-modified FET, as shown in Fig. 3. The PBA-modified FET was then tested for its ability to directly capture the glycan component SA present on the cell surface. As for proof-of-principle, erythrocyte was investigated, for which alternations of the surface SA content have been reported in diabetes mellitus. Fig. 4 display monitorings of the VT changes on adding suspensions of (a) native rabbit erythrocytes and (b) those with enzymatically decreased surface SA (20% remains) with step-wise increase of the concentration onto the PBA-modified FET. The slopes of the VT changes obtained in Fig. 4 reflect the altered amount of SA per cell. This suggests that once a cell number-VT calibration line is determined for a healthy phenotype, the altered SA expression level on the erythrocyte can directly be monitored in a real-time manner, simply by placing the known-count living cell suspensions onto the device. We demonstrated that a PBA-modified electrode with properly controlled pKa could differentiate the degree of tumor metastasis through perception of the cell membranes SA. The technique can be readily extended to other primary vs tissue systems if their cell number-VT calibrations are predetermined. Such a capability may serve as a remarkably easy and quantitative adjunct to histological evaluation of tumor malignancy and metastatic potential during intraor postoperative diagnoses. Alternations of sialic acid (SA) contents on cell surface glycan chains have been implicated in numerous normal and pathological processes including developments, differentiations, diabetes and tumor metastasis. Techniques to conveniently monitor cell surface SA therefore have great relevance to handy ways of cytology. Ordinarily, cell surface SA density is assessed via multiple enzymatic and labelling procedures, which, however, involve severely invasive, in many cases lethal procedures. In the context of practical applications, these are very unlikely to provide a feasible diagnostic platform. The method developed using bio-transistors has been achieved only by placing the known-count living cell suspensions on the electrode without any enzymatic, labeling and lethal procedures that are unavoidable in any other existing determination methods.

Biochemical Responses of Nano-Pillar Gate Field-Effect Devices

We have developed biochemical field effect devices with nano-pillar array structure on the gate. The pH responses of the field effect device with nano-pillar structure were nonlinear. It seems that the plasma treatment of the Si3N4 surface as well as a mixed composition of Si3N4 and SiO2 may play an important role for the nonlinearity of the pH response. Oligonucleotide probes with fluorescent label were immobilized on the surface of the nano-pillar gate structure. The fluorescent intensity of the device with nano-pillar structure was stronger than that of the flat gate device. The increase of the fluorescent intensity for the nano-pillar device could be explained by the increase of the number of immobilized oligonucreotide probes because of the larger surface area. We can therefore expect highly sensitive detection of biomolecules with the nano-pillar gate field effect devices.