JP5467312B2 - Biosensor, detection method of biological material using biosensor, and kit thereof - Google Patents

Description

  The present invention relates to a biosensor, and more particularly to a biosensor that can be used for detection and analysis of sialic acid derived from a cell sugar chain.

  In recent years, analysis of sugar chains in cells has attracted attention as post-genome research following post-genome research centered on structural and functional analysis of genes and proteins. Sugar chains are deeply involved in cell-to-cell interactions, and their structures change to reflect “cell states” such as various diseases, abnormalities in immunity and development, and aging, so they are often described as “cell faces”. Is done. In addition, since many biomarkers typified by tumor markers are thought to be sugar chains, it is urgent to search for functional sugar chains closely related to biological functions such as the occurrence of cancer.

  Furthermore, also in the medical field, in order to determine a patient's medical policy rapidly, it is calculated | required to analyze the state of many target cells rapidly by analyzing the sugar chain as a biomarker.

  As a means for analyzing such sugar chains, conventionally, a method of labeling sugar chains in cells by UV, fluorescence or the like has been employed. For example, in Non-Patent Document 1, sialic acid derived from a glycan released by sialidase or acid is derivatized with 1,2-diamine-4,5-methylenedibenzone (DMB), which is a fluorescent reagent, and high performance liquid chromatography (HPLC ) Is used to quantify sialic acid. In addition, a method has been developed in which sialic acid is quantified by converting the liberated sialic acid to N-acyl mannosamine by sialic acid aldolase and hydrolyzing the N-acyl mannosamine (patent). Reference 1).

JP 2000-333698 A

Analytical Biochemistry 179, 162-166 (1989)

  By the way, in the sugar chain analysis method for labeling sugar chains as described above, in order to label a target cell, a label-dedicated device and a facility for labeling must be prepared. In many cases, such devices and facilities are large and expensive, so that an economic burden is great for performing a sugar chain analysis.

  In addition, it is necessary to go through the two-step process of first labeling the cells to be analyzed and then performing the analysis, which makes it impossible to meet the high-speed and high-throughput glycan analysis required in medical settings. .

  The present invention has been made in view of such circumstances, and is a non-invasive and easy-to-use real-time cell that does not use a label such as fluorescence, which has been impossible with conventional sugar chain analysis methods. It is an object of the present invention to provide a diagnostic tool and a method for detecting a sugar chain of a target cell using the diagnostic tool.

  According to a first main aspect of the present invention, the biosensor has a detection surface, and the detection surface is coated with a phenylboronic acid group that binds to sialic acid derived from a cell sugar chain. Is provided.

  According to such a configuration, since a physical change can be caused on the detection surface by binding with the sialic acid derived from the cell sugar chain, for example, a field effect transistor, an optical waveguide member, or a piezoelectric member is provided. By utilizing this, sialic acid can be detected.

  According to one embodiment of the present invention, the biosensor includes a metal layer, a self-assembled monolayer of organic molecules formed by chemisorption on the metal layer, and a self-assembled monolayer of the self-assembled monolayer. It has a phenylboronic acid group which is introduced on the surface opposite to the metal layer and constitutes the detection surface.

  Here, the biosensor has a detection medium for detecting any one or more changes in physical characteristics including optical characteristics, vibration characteristics, and electrical characteristics due to sialic acid binding to the detection surface. The detection surface is preferably formed on the detection medium.

  For example, the detection medium may be a gate of a field effect transistor. In this case, the biosensor includes a metal layer provided on the gate and organic molecules formed on the metal layer by chemisorption. It is preferable to have a self-assembled monolayer and a phenylboronic acid group that is introduced on the surface of the self-assembled monolayer opposite to the metal layer and constitutes the detection surface.

  Similarly, when the detection medium is a gate of a field effect transistor, the biosensor is provided with a first metal layer provided on the gate and spaced apart from the gate, and the first metal A second metal layer connected to the layer through conductive wiring, a self-assembled monolayer of organic molecules formed on the second metal layer by chemical adsorption, and the self-assembled monolayer It may have a phenyl boronic acid introduced on the surface opposite to the substrate and constituting the detection surface.

  The detection medium may be an optical waveguide member or a piezoelectric member having the detection surface formed on the outer surface thereof. In this case, when the sialic acid is bonded to the phenylboronic acid group, the light guide member It is preferable that the optical characteristic and / or the vibration characteristic is changed at the boundary between the wave member or the piezoelectric member and the detection surface.

  Further, according to a second main aspect of the present invention, there is provided a method for detecting sialic acid derived from a cell sugar chain, comprising a step of preparing a test sample containing a cell sugar chain, and a detection surface, Preparing a biosensor in which the detection surface is coated with a phenylboronic acid group that binds to sialic acid in the test sample, contacting the prepared test sample with the biosensor, and the test sample And a step of detecting a physical signal including optical characteristics, vibration characteristics, and electrical characteristics that change as a result of a reaction between the sialic acid in the group and the phenylboronic acid group. Provided.

  According to such a configuration, a method for detecting sialic acid derived from a cell sugar chain using the biosensor according to the first aspect described above is provided.

  According to one embodiment of the present invention, in the method according to the second aspect, the biosensor includes a metal layer and a self-assembled monolayer of organic molecules formed on the metal layer by chemical adsorption. And a phenylboronic acid group that is introduced on the surface of the self-assembled monolayer opposite to the metal layer and constitutes the detection surface.

  Here, in this method, the detecting step is for detecting any one or more changes in physical characteristics including optical characteristics, vibration characteristics, and electrical characteristics due to sialic acid binding to the detection surface. The detection is preferably performed via a detection medium, and the detection surface is preferably formed on the detection medium.

  For example, the detecting step may be performed through a gate of a field effect transistor as the detection medium. In this case, the biosensor includes a metal layer provided on the gate, and a metal layer on the metal layer. A self-assembled monolayer of organic molecules formed by chemisorption, and a phenylboronic acid group that is introduced on the surface opposite to the metal layer of the self-assembled monolayer and constitutes the detection surface It is preferable.

  Similarly, when the detecting step is performed through a gate of a field effect transistor as the detection medium, the biosensor is spaced apart from the first metal layer provided on the gate and the gate. And a second metal layer connected to the first metal layer via a conductive wiring, and a self-assembled monolayer of organic molecules formed by chemical adsorption on the second metal layer And a phenylboronic acid introduced on the surface of the self-assembled monolayer opposite to the substrate and constituting the detection surface.

  In this method, the detecting step is performed via a detection medium that is an optical waveguide member or a piezoelectric member in which the detection surface is formed on the outer surface, and the sialic acid is bonded to the phenylboronic acid group. It is preferable to detect a change in optical characteristics and / or vibration characteristics at a boundary portion between the optical waveguide member or piezoelectric member and the detection surface.

  According to a third main aspect of the present invention, there is provided a detection kit for detecting sialic acid derived from a cell sugar chain, comprising the biosensor according to the first main aspect described above.

  According to such a configuration, a detection kit for detecting sialic acid derived from a cell sugar chain using the biosensor according to the first aspect described above is provided.

  According to a fourth main aspect of the present invention, there is provided a method for determining the presence or absence of disease cells in a sample derived from a living body, the step of collecting a test sample containing a cell sugar chain from the living body, A step of providing a biosensor having a detection surface, the detection surface being coated with a phenylboronic acid group that binds to sialic acid in the test sample, and bringing the collected test sample into contact with the biosensor A step of detecting a physical signal including a light characteristic, a vibration characteristic, and an electric characteristic that are changed by a reaction between the sialic acid in the test sample and the phenylboronic acid group, and the detected physical signal. And a step of comparing the physical signal obtained when subjecting the normal cell to the biosensor, and a process for determining whether the test sample is a disease cell based on the comparison result And having bets, a method is provided.

  According to such a configuration, there is provided a method for determining the presence or absence of disease cells in a biological sample using the biosensor according to the first aspect described above.

  In this case, the diseases are diabetes, lung cancer, esophageal cancer, stomach cancer, rectal cancer, liver cancer, pancreatic cancer, kidney cancer, skin cancer, bladder cancer, breast cancer, uterine cancer, vaginal cancer, leukemia, osteosarcoma, brain tumor, spinal cord It is preferably one selected from the group consisting of tumor, neuroblastoma, thyroid cancer, prostate cancer and vulvar cancer.

In the method according to the fourth main aspect, the step of determining includes
When the value obtained from the value related to the detected physical signal / value related to the physical signal in the normal cell is 0.9 or less, or 1.1 or more, it is determined that the cell is a diseased cell. Is preferred.

  According to one embodiment of the present invention, in the method according to the third aspect, as in the method according to the second aspect, the biosensor includes a metal layer and a chemical layer on the metal layer. Having a self-assembled monolayer of organic molecules formed by adsorption, and a phenylboronic acid group constituting the detection surface introduced to the surface of the self-assembled monolayer opposite to the metal layer It is.

  Here, in this method, the detecting step is for detecting any one or more changes in physical characteristics including optical characteristics, vibration characteristics, and electrical characteristics due to sialic acid binding to the detection surface. The detection is preferably performed via a detection medium, and the detection surface is preferably formed on the detection medium.

  For example, the detecting step may be performed through a gate of a field effect transistor as the detection medium. In this case, the biosensor includes a metal layer provided on the gate, and a metal layer on the metal layer. A self-assembled monolayer of organic molecules formed by chemisorption, and a phenylboronic acid group that is introduced on the surface opposite to the metal layer of the self-assembled monolayer and constitutes the detection surface It is preferable.

  Similarly, when the detecting step is performed through a gate of a field effect transistor as the detection medium, the biosensor is spaced apart from the first metal layer provided on the gate and the gate. And a second metal layer connected to the first metal layer via a conductive wiring, and a self-assembled monolayer of organic molecules formed by chemical adsorption on the second metal layer And a phenylboronic acid introduced on the surface of the self-assembled monolayer opposite to the substrate and constituting the detection surface.

  In this method, the detecting step is performed via a detection medium that is an optical waveguide member or a piezoelectric member in which the detection surface is formed on the outer surface, and the sialic acid is bonded to the phenylboronic acid group. It is preferable to detect a change in optical characteristics and / or vibration characteristics at a boundary portion between the optical waveguide member or piezoelectric member and the detection surface.

  It should be noted that the features of the present invention other than those described above and the remarkable actions and effects will become clear to those skilled in the art with reference to the following embodiments and drawings of the present invention.

FIG. 1 is an overall schematic diagram showing a detection system using a biosensor according to a first embodiment of the present invention. FIG. 2 is a schematic diagram showing the binding of sialic acid and phenylboronic acid groups on the detection surface of the biosensor according to the first embodiment of the present invention. FIG. 3 is a diagram showing a chemical reaction formula of a reaction between sialic acid and a phenylboronic acid group. FIG. 4 is a graph showing free sialic acid detected by the biosensor according to the first embodiment of the present invention. FIG. 5 is a graph showing rabbit erythrocyte surface sialic acid detected by the biosensor according to the first embodiment of the present invention. FIG. 6 is a view showing the production of a lung metastatic cancer model mouse. FIG. 7 is a view showing lung metastatic cancer model mice having different degrees of metastasis. FIG. 8 is a graph showing the relationship between the threshold voltage obtained using the biosensor according to the present invention and the degree of cancer metastasis. FIG. 9 is a schematic view showing a biosensor according to the second embodiment of the present invention. FIG. 10 is an overall schematic diagram showing a detection system using a biosensor according to the third embodiment of the present invention. FIG. 11 is an overall schematic diagram showing a detection system using a biosensor according to the fourth embodiment of the present invention.

Hereinafter, an embodiment and an example of the present invention will be described with reference to the accompanying drawings.
(First embodiment)
First, a first embodiment of the present invention will be described with reference to FIGS.

  The first embodiment is an example in which the biosensor of the present invention is configured using a field effect transistor (FET).

FIG. 1 is an overall schematic diagram showing a detection system 1 using a biosensor.
The detection system 1 includes, for example, a biosensor unit 3, a sensor driving circuit unit 4 that drives the biosensor unit 3 by giving a signal to the printed mounting board 2, and a power supply unit 5 that supplies power to the entire board. And a detection circuit unit 6 that processes the output from the biosensor unit 3 and outputs a detection signal, and an output interface (output IF) unit 7 for outputting the detection signal to the outside. And integrated.

  The biosensor 8 provided in the biosensor unit 3 includes a field effect transistor (FET) including a source 10 and a drain 11 formed on the surface of a semiconductor substrate 9 and a gate 12 formed on the semiconductor substrate 9. It has a structure. The surface of the gate 12 is covered with a phenylboronic acid group 14 via a metal layer 13 to constitute the detection surface 16 of the present invention. This detection surface 16 is surrounded by the measurement cell wall 15, thereby demarcating the detection region.

  According to such a biosensor 8, a voltage is applied to the gate 12 using, for example, the reference electrode 17, and in this state, a sialic acid sample derived from a cell sugar chain (cell itself or cell-derived) is applied to the detection surface 16. When the sialic acid in the sample is bonded to the phenylboronic acid group 14 coated on the detection surface 16, the value of the current flowing between the source 10 and the drain 11 changes. This change in current is sampled by an ammeter 18, processed by the detection circuit unit 6, and extracted from the output IF unit 7 as a predetermined detection signal.

  In this example, the biosensor unit 3 is illustrated as including the power source 19, but actually, the biosensor unit 3 is driven by the sensor driving circuit unit 4 mounted on the printed mounting board 2. A power supply unit 5 for supplying power to the sensor drive circuit unit 4 is also mounted in the same manner.

  In this embodiment, all the components including the biosensor unit 3 can be integrated on a single printed mounting board 2, and the sialic acid derived from the cell sugar chain has a very small and simple configuration. It is possible to obtain a biosensor and a biosensor system that can detect

  In addition, the field effect transistor (FET) is a completely non-invasive and non-labeling measurement method that detects the molecular intrinsic charge captured on the detection surface 16 in synchronization with the change in transistor characteristics, and is a real-time measurement, It covers the major requirements required for high-throughput systems such as low cost because it does not require an optical system, which is advantageous for downsizing, and easy high-density and super-parallelization by semiconductor processing technology.

Next, the configuration of the detection surface 16 will be described in detail with reference to FIG.
FIG. 2 is a schematic diagram showing the binding between sialic acid 21 derived from cell sugar chain 20 and phenylboronic acid group 14 attached to detection surface 16.

  As described above, according to the present invention, the detection surface 16 of the biosensor is coated with the phenylboronic acid group 14 that binds to the sialic acid 21 derived from the cell sugar chain 20, and the sialic acid 21 and the phenylboronic acid group 14 are bonded. The gist is to detect the physical change of the detection surface caused by the above, that is, the generation of the negative charge 22 in FIG.

  In order to form such a detection surface 16, in this embodiment, an Au vapor deposition thin film as a metal layer 13 is formed on the gate 12, and the self-organization of organic molecules formed by chemical adsorption on the vapor deposition thin film. A monomolecular film (SAM) 23 is formed. A phenylboronic acid group 14 is introduced into the end of the self-assembled monolayer 23 opposite to the metal layer 13 by a condensation reaction or the like.

  According to such a configuration using the self-assembled monolayer 23, the detection surface 16 where the bond between the phenylboronic acid group 14 and the sialic acid 21 occurs can be configured at a position very close to the gate 12 of the FET. . Thus, the physical change of the detection surface 16 due to the combination of sialic acid-phenylboronic acid groups in the FET, in this example, the change of the charge 22 of the detection surface 16 can be detected accurately and easily.

  FIG. 3 is a chemical reaction formula showing that a negative charge is generated by the bond between the sialic acid and the phenylboronic acid group as described above. In this example, a bond between 3-propionamidophenylboronic acid and sialic acid is shown. In an aqueous solution at pH 7.4, the bond between boronic acid and sialic acid is dominant over the bond with other sugar compounds (H. Otsuka et al., J. Am. Chem. Soc. 125). , 3493, (2003)). Here, sialic acid has a negative charge derived from a carboxyl group, and in the biosensor 8 according to the present invention, this negative charge is detected by the FET on the detection surface 16.

  Here, “sialic acid” in the present specification refers to a family name having the following chemical formula and generically referring to substances in which the amino group or hydroxy group of neuraminic acid is substituted. .

  Sialic acid is a molecule that is most frequently present in sugar chains and abundantly at the sugar chain ends, and its density and distribution are related to cell diseases (cancer, metastasis, lifestyle-related diseases, autoimmune diseases) and outbreaks, It strongly correlates with various cellular phenomena such as differentiation. Several derivatives of sugar chain sialic acid are known, but it has been reported that their density and composition differ significantly in cancerous cells (Hakomori, S. Cancer Res. 1985, 45, 2405-2414). ). More particularly, the strength of the interaction between these sialic acid derivatives and phenylboronic acid can be controlled by the phenyl ring substituent structure of the phenylboronic acid group. Most recently, examples of phenylboronic acid molecular designs that have acquired recognition ability for specific sugar chain structures by introducing special substituents have also been reported (Dowlut, M .; Hall, DGJ Am. Chem). Soc. 2005, 128, 4226-4227).

  Therefore, by using the biosensor of the present invention, it is possible to easily detect, prevent and treat cancer, metastasis, lifestyle-related diseases and autoimmune diseases by a non-invasive and non-labeling method.

  The sialic acid may be present in a cell sugar chain or may be a sialic acid present in a sugar chain sample collected from a cell by an enzyme or the like.

  In addition, the “phenylboronic acid group” used in the present specification refers to the following chemical formula.

  In this embodiment, an example of 3-acrylamidophenylbornonic acid is shown as the phenylboronic acid group constituting the detection surface, but any compound may be used as long as it has the above structural formula.

  As the FET, any FET used for a conventional biosensor or a complementary metal oxide semiconductor (CMOS) element can be used, and any of n-MOS and p-MOS can be used. is there. This FET is composed of a source for supplying carriers (free electrons or holes), a drain to which the carriers supplied by the source reach, and a gate for controlling the flow of carriers between the source and drain. If it is what.

The metal layer formed on the gate may be any material into which the SAM and the phenylboronic acid group can be introduced, but preferably, Au, SiO ₂ , TiO ₂ , and It is preferably made of any one material selected from the group consisting of Al ₂ O ₃ .

  Hereinafter, examples according to the first embodiment will be described.

(experimental method)
A gold-deposited substrate was used as the gate 12 of the FET, and a self-assembled monolayer (SAM) by 10-carboxy-1-decanethiol was previously formed thereon. A “sialic acid recognition transistor”, which is a biosensor, was created by introducing 3-acrylamidophenylboronic acid into the carboxyl group terminal of SAM by a condensation reaction. While adding various monosaccharides and rabbit erythrocytes (untreated and sialidase treatment) at a predetermined concentration (pH 7.4 PBS-0.9% NaCl) on the prepared gate 12, the gate surface potential was measured using a real-time FET measuring device. The change was observed.

(result)
The free sialic acid detection result by the created biosensor is shown in FIG. The gate surface potential changed significantly in the region where the added sialic acid concentration was 300 to 900 μM, and was saturated at a concentration of 900 μM or more. The potential shift in the negative direction observed in FIG. 4 corresponds to the carboxyl group in sialic acid. None of the measurements (confirmed up to 10 mM) were detected in the measurement using a gate without boronic acid introduced as a control and in the measurement performed by adding glucose, galactose and mannose as control monosaccharides. . As described above, it was revealed that the fabricated transistor specifically senses free sialic acid with a sensitivity of about 100 μM.

  FIG. 5 shows the relationship between red blood cell concentration and potential change when rabbit red blood cells are continuously seeded on the gate 12 of the FET. When the sialic acid unit was partially cleaved by sialidase treatment, a small potential change was observed compared to untreated. This is presumably because the adhesion of the erythrocytes to the gate surface via a specific bond with the phenylboronic acid group decreases due to the removal of sialic acid at the sugar chain end.

(Conclusion)
A "sialic acid recognition transistor" was created, and the difference in sugar chain sialic acid density on the cell surface was successfully detected noninvasively and unlabeled. By considering sialic acid as a “cell face” and using it as a target for sugar chain profiling (feature extraction), it is possible to develop a simpler, cheaper, and faster cytodiagnosis technique than before.

(Method Using First Embodiment)
In the above example, it has been explained that a method for detecting sialic acid in a specific cell can be provided. However, by using the FET according to the present embodiment, a method for further determining the presence or absence of a specific disease cell is provided. Can be provided. In this case, similarly to the method for detecting sialic acid, it is determined whether or not the cell suffers from a specific disease by seeding the cell having a sugar chain on the detection surface of the FET according to the present embodiment. To do.

  Here, the above determination is performed by utilizing the fact that the amount of sialic acid in the sugar chain on the cell surface affected by a specific disease is smaller or larger than the sugar chain on the surface of a normal cell. For example, it is known that the amount of sialic acid on the cell surface is reduced in erythrocytes of patients suffering from certain types of diabetes. It is also known that sugar chain modification mutations occur with canceration, and it is known that the amount of sialic acid is increased in the sugar chains of the diseased cells of patients suffering from certain types of cancer . By using this phenomenon, the amount of sialic acid in normal cells is compared with the amount of sialic acid in diseased cells. If it is decreased, it can be determined as a diseased cell.

  When comparing the amount of sialic acid in normal cells and the amount of sialic acid in diseased cells, from the quantitative relationship, one measurement value is simply divided by the other measurement value, and the resulting value is 0. When the number is 9 or less, or 1.1 or more, the cell can be determined to be a disease cell. Preferably, a disease cell is determined when the value of the result is 0.7 or less, or 1.3 or more. More preferably, when the value of the result is 0.5 or less, or 1.5 or more, it is determined that the cell is a diseased cell. In this case, the measurement value may be standardized as necessary before the division.

  As described above, in addition to being able to make a determination based on the structural change of cell sugar chain modification accompanying a disease, in addition, for example, a fluorescent substance such as luciferase or GFP is expressed in a disease cell. It may be configured to check the expression (increase or decrease) of sialic acid supplementarily by comparing with the expression frequency.

  In the method for determining the presence or absence of a specific disease cell described above, the disease is any disease as long as the cell sugar chain modification is known to be mutated or structurally changed by the disease. There may be. In particular, it is preferable that the disease is known to change the amount of sialic acid in the cell sugar chain depending on the disease. For example, as described above, it is known that the amount of sialic acid in the cell sugar chain increases or decreases in diabetes and certain types of cancer. In addition, it can also be applied to leukemia, osteosarcoma, neuroblastoma, etc. in which abnormal cell proliferation ability is observed.

  As a method for determining the presence or absence of a specific disease cell, the same FET as that used in the method for detecting sialic acid in a specific cell can be used. For example, as shown in FIG. 1 described above, in the FET used in the above two methods, the surface of the gate 12 is coated with phenylboronic acid groups 14 via the metal layer 13, and the detection surface 16 is a measurement cell. Surrounded by a wall 15, the detection region is thereby partitioned.

  Furthermore, the degree of metastasis of cancer can also be measured by using the above-described method for determining the presence or absence of a specific disease cell. In this case, the degree of metastasis can be determined by calculating the cells determined to be disease cells per whole cell collected from a living body as a sample. As used herein, “degree of metastasis” refers to the proportion of cancer cells in a certain organ or site, and is derived from a cancer that exists in an organ or site other than the organ or site for which the degree of metastasis is measured. Is how much the cancer has metastasized.

  In the above-described method, it is preferable to remove the aggregated cell mass by filtering the cells with a cell strainer before seeding the FETs according to the present embodiment. By filtration with this cell strainer, each cell to be tested is separated, and the amount of sialic acid in the cell can be detected more accurately.

  Moreover, different types of cells can be used for each disease as cells to be tested. In this case, the selection criteria for the cells are, for example, cells that can appropriately reflect the disease state in each disease, and the selected cells are, for example, erythrocytes in whole blood if diabetic. In the case of lung cancer, lung cells may be mentioned.

  Below, the experiment example at the time of determining the presence or absence of a specific disease cell using FET which concerns on this embodiment is demonstrated using FIGS.

(Experimental method and result relating to the method of determining the presence or absence of a specific disease cell)
First, as a metastatic luciferase-expressing mouse melanoma, a metastatic murine melanoma expressing luciferase (B16-F10-Luc-G5) was prepared, and subcutaneous injection (10 ⁶ cells) in the tail of a healthy mouse (BALB / c nu / nu). / ML). The melanoma was made to metastasize to the lung.

  FIG. 6 is a diagram showing that a lung metastasis cancer model mouse was actually prepared. A lung metastatic cancer model mouse subcutaneously injected with metastatic murine melanoma expressing luciferase (B16-F10-Luc-G5) was treated with luciferin, and chemiluminescence was quantified using IVIS imaging system (Xenogen). The photograph shown in the left part of this figure was taken using an IVIS imaging system of a lung metastatic cancer model mouse subcutaneously injected with a metastatic melanoma expressing luciferase (B16-F10-Luc-G5) at the tail. As shown in this photograph, melanoma injected subcutaneously in the tail can metastasize to the lungs. Further, the graph shown on the right side of this figure is a graph in which the horizontal axis represents the number of cells and the vertical axis represents the amount of luminescence. As shown in this graph, the number of cells also increases in proportion to the amount of luminescence.

Subsequently, lung cells were collected from the lung metastatic cancer model mouse and filtered with a cell strainer to remove cell clumps. Thereafter, the threshold voltage value change (ΔV _T , mV) was measured at various cell numbers using the sialic acid detection FET (biosensor) according to the present invention.

FIG. 7 shows bioluminescence images of specimens obtained by transferring luciferase-expressing mouse melanoma (B16-F10-Luc-G5) to mouse lungs at different degrees of metastasis (0%, 15%, 30%). is a graph showing gross image, and threshold voltage changes in a variety of cell number (ΔV _T, mV). As shown in this figure, as the number of days after subcutaneous injection passes, the amount of luminescence showing metastasis to the lung increases, and the threshold voltage change (ΔV _T , mV) in various cell numbers also increases. .

  FIG. 8 is a graph showing the relationship between the threshold value voltage obtained by using the sialic acid detection FET according to the present invention and the degree of cancer metastasis. FIG. 8A is a threshold voltage obtained using the sialic acid detection FET according to the present invention, and FIG. 8B is a graph showing the amount of sialic acid in a lung metastatic cancer model mouse obtained by a conventional method. As shown in FIG. 8A, it can be seen that the threshold voltage increases as the degree of metastasis to the lung increases in various cell numbers. This indicates that the amount of sialic acid in the cell increases as the degree of metastasis to the lung increases. In addition, the threshold voltage increases as the number of cells increases when compared at the same degree of metastasis. This result shows that the amount of sialic acid increases as the number of cells increases. FIG. 8B shows the result of quantifying sialic acid by a conventional method using the same experimental system. This graph proves that the data for various cell numbers are in good agreement with those in FIG. 8A, and that the sialic acid detection FET according to the present invention can accurately measure the amount of sialic acid in the cell sugar chain. Similarly, these results show that it is possible to determine whether a specific cell is a disease cell by using the sialic acid detection FET according to the present invention.

(Conclusion)
As described above, the sialic acid detection FET according to the present invention detects sugar chain sialic acid on the cell surface, and thereby can determine the presence or absence of a disease in a specific cell. Moreover, the degree of metastasis of cancer cells to a specific tissue or site can also be measured by calculating the number of cells determined to be diseased cells per total collected cell.

(Modification of this embodiment)
In addition, this invention is not limited to one Embodiment mentioned above, A various deformation | transformation is possible in the range which does not change the summary of invention.

(Second Embodiment)
For example, in the first embodiment described above, the configuration in which the detection surface 16 covered with the phenylboronic acid group 14 is directly formed on the gate 12 has been described. However, the detection surface 16 is a detection medium as the gate 12. It may be formed away from.

  FIG. 9 shows a schematic diagram of such an example. That is, in this example, the first metal layer 25 is provided on the gate, and the detection surface 16 as described above is formed on the second metal layer 26 connected to the first metal layer 25 via the wiring. Formed. Configurations other than those described here are the same as those in the first embodiment.

Even with such a configuration, it is possible to obtain the same effect as that of the first embodiment.
(Third and fourth embodiments)

  In the first and second embodiments described above, the FET is used as an example, but the present invention is not limited to this. In the third and fourth embodiments, surface plasmon resonance (SPR) and Quartz Crystal Microbalance (QCM) are used as other examples.

  In the third and fourth embodiments described below, for the sake of convenience of explanation, the same reference numerals are given to the same configurations as those of the first embodiment, and the description thereof is simplified.

FIG. 10 is a schematic diagram showing an example in which the SPR is used as the third embodiment.
SPR means that laser light L is incident on the metal layer 13 from one end face of the solid phase substrate 28 at an angle greater than the critical angle, whereby the metal layer 13 and the sample coated on the surface of the metal layer 13 are It generates surface plasmons at the boundary surface.

  In this example, the metal layer 13 provided on the solid phase substrate 28 is coated with the phenylboronic acid group 14, and the laser beam L is emitted from one end face of the solid phase substrate 28 by the measuring light irradiation means 29 at an angle greater than the critical angle. Is incident. Then, the reflected light intensity change caused by this is detected by the reflected light measuring means 30, the refractive index of the phenylboronic acid group-coated surface is obtained from the attenuation of the optical intensity, and the test sample, that is, the sialic acid derived from the cell sugar chain Analyze.

  When SPR is used as the biosensor 8 according to the present invention, an optical system using an optical fiber, a prism, a diffraction grating, an optical waveguide, or the like can be used as a method for causing surface plasmon resonance. As the phase substrate material, glass, polymer resin, plastic or the like can be used. The object of measurement in surface plasmon resonance may be the wavelength of light or the angle of incident reflection, LED, LD, white light, etc. as the light source, CCD, PD, light as the reflected light measuring means 30 A position sensor or the like can be used.

FIG. 11 is a schematic diagram showing an example in which the QCM is used as the fourth embodiment.
The QCM sensor 33 is a small and highly sensitive mass detector that can measure the mass of a trace substance adsorbed or bonded to the surface of the crystal unit 34 from the change in the resonance frequency of the crystal unit 34. In this detection system using the QCM sensor 33, the QCM sensor 33 is arranged so that the electrode surface 35 coated with the phenylboronic acid group 14 adheres to the sialic acid sample (solution or gas). Then, a change in electrical characteristics due to the bonding between the phenylboronic acid group 14 and the sialic acid sample, for example, a change in the oscillation frequency of the oscillation circuit unit 36 is measured by the measurement unit 37.

  When the QCM sensor is used for sialic acid detection, it is preferable to use as the solid phase a structure in which the phenylboronic acid group 14 that binds to sialic acid is formed on the electrode surface 34 of the crystal resonator 34. This crystal oscillator 34 is placed in a container filled with a buffer solution, and then a sample containing sialic acid (cell or a sugar chain sample obtained from the cell) is added. Sialic acid binds to the phenylboronic acid group 14 formed in the solid phase on the electrode surface 35 of the crystal resonator 34 and becomes a mass load, thereby reducing the resonance frequency of the crystal resonator 34. The advantage of the QCM sensor is that a calibration curve is not required because the mass change amount can be obtained directly from the frequency change amount.

DESCRIPTION OF SYMBOLS 1 ... Detection system 2 ... Print mounting board 3 ... Biosensor part 4 ... Sensor drive circuit part 5 ... Power feeding part 6 ... Detection circuit part 7 ... Output interface part 8 ... Biosensor 9 ... Semiconductor substrate 10 ... Source 11 ... Drain 12 ... Gate 13 ... Metal layer 14 ... Phenylboronic acid group 15 ... Measurement cell wall 16 ... Detection surface 17 ... Reference electrode 18 ... Ammeter 19 ... Power source 20 ... Sugar chain 21 ... Sialic acid 22 ... Negative charge 23 ... Self-assembled monomolecule Membrane (SAM)
DESCRIPTION OF SYMBOLS 25 ... 1st metal layer 26 ... 2nd metal layer 28 ... Solid phase substrate 29 ... Measurement light irradiation means 30 ... Reflection light measurement means 33 ... QCM sensor 34 ... Crystal oscillator 35 ... Electrode surface 36 ... Oscillation circuit part 37 ... Measurement part L ... Laser light

Claims (16)

A biosensor characterized in that it has a detection surface, and this detection surface is coated with a phenylboronic acid group that binds to sialic acid derived from a cell sugar chain,
A metal layer,
A self-assembled monolayer of organic molecules formed by chemisorption on the metal layer;
The self-assembled monolayer has a phenylboronic acid group introduced on the surface opposite to the metal layer and constituting the detection surface,
The biosensor according to claim 1, wherein the phenylboronic acid group bonded to the self-assembled monolayer has a meta-amide substituent structure. The biosensor according to claim 1, wherein
A detection medium for detecting a change in one or more of physical characteristics including optical characteristics, vibration characteristics, and electrical characteristics due to sialic acid binding to the detection surface;
The detection surface is formed on the detection medium. A biosensor. The biosensor according to claim 2, wherein
The biosensor according to claim 1, wherein the detection medium is a gate of a field effect transistor. The biosensor according to claim 3, wherein
The biosensor, wherein the metal layer is provided on the gate. The biosensor according to claim 3, wherein
The metal layer is
A first metal layer provided on the gate;
A second metal layer provided apart from the gate and connected to the first metal layer through a conductive wiring;
The biosensor according to claim 1, wherein the detection surface is formed on the second metal layer. The biosensor according to claim 2, wherein
The detection medium is an optical waveguide member or a piezoelectric member in which the detection surface is formed on an outer surface thereof,
When the sialic acid is bonded to the phenylboronic acid group, optical characteristics and / or vibration characteristics at a boundary portion between the optical waveguide member or the piezoelectric member and a detection surface are changed. Biosensor. A method for detecting sialic acid derived from a cell sugar chain,
Preparing a test sample containing cellular sugar chains;
Providing a biosensor having a detection surface, the detection surface being coated with a phenylboronic acid group that binds to sialic acid in the test sample;
Contacting the prepared test sample with the biosensor;
Detecting physical signals including light characteristics, vibration characteristics, and electrical characteristics that change as a result of a reaction between the sialic acid in the test sample and the phenylboronic acid group;
A method comprising:
The biosensor is
A metal layer,
A self-assembled monolayer of organic molecules formed by chemisorption on the metal layer;
The self-assembled monolayer has a phenylboronic acid group introduced on the surface opposite to the metal layer and constituting the detection surface,
The method wherein the phenylboronic acid group has a meta-amide substituent structure. The method of claim 7, wherein
The detecting step includes
Carried out via a detection medium for detecting any one or more changes in physical characteristics including optical characteristics, vibration characteristics, and electrical characteristics due to sialic acid binding to the detection surface;
The method, wherein the detection surface is formed on the detection medium. The method of claim 8, wherein
The detecting step is performed through a gate of a field effect transistor as the detection medium. The method of claim 9, wherein
The biosensor is
A method characterized in that a metal layer is provided on the gate. The method of claim 9, wherein
This biosensor
A first metal layer provided on the gate;
A second metal layer provided apart from the gate and connected to the first metal layer via a conductive wiring;
On the second metal layer, the self-assembled monolayer of organic molecules formed by chemisorption,
And a phenylboronic acid group which is introduced on a surface opposite to the substrate of the self-assembled monolayer and constitutes the detection surface. The method of claim 8, wherein
The detecting step is performed via a detection medium that is an optical waveguide member or a piezoelectric member in which the detection surface is formed on an outer surface,
When the sialic acid is bonded to the phenylboronic acid group, it detects a change in optical characteristics and / or vibration characteristics at a boundary portion between the optical waveguide member or piezoelectric member and a detection surface. how to.   A detection kit for detecting sialic acid derived from a cell sugar chain, comprising the biosensor according to claim 1. A method for determining the presence or absence of diseased cells in a biological sample,
Preparing a test sample containing a sugar chain collected from a living body ;
Providing a biosensor having a detection surface, the detection surface being coated with a phenylboronic acid group that binds to sialic acid in the test sample;
Contacting the collected test sample with the biosensor;
Detecting physical signals including light characteristics, vibration characteristics, and electrical characteristics that change as a result of a reaction between the sialic acid in the test sample and the phenylboronic acid group;
Comparing the detected physical signal with a physical signal obtained when subjecting a normal cell to the biosensor;
And determining whether the test sample is a disease cell based on the comparison result,
The biosensor is
A metal layer,
A self-assembled monolayer of organic molecules formed by chemisorption on the metal layer;
The self-assembled monolayer has a phenylboronic acid group introduced on the surface opposite to the metal layer and constituting the detection surface,
The method wherein the phenylboronic acid group has a meta-amide substituent structure. The method of claim 14, wherein
The diseases are diabetes, lung cancer, esophageal cancer, stomach cancer, rectal cancer, liver cancer, pancreatic cancer, kidney cancer, skin cancer, bladder cancer, breast cancer, uterine cancer, vaginal cancer, leukemia, osteosarcoma, brain tumor, spinal cord tumor, nerve A method which is selected from the group consisting of blastoma, thyroid cancer, prostate cancer, vulvar cancer. The method of claim 14, wherein
The step of determining includes
When the value obtained by the value relating to the detected physical signal / the value relating to the physical signal in the normal cell is 0.9 or less, or 1.1 or more, it is determined that the cell is a disease cell. Method.

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