Abstract:
One molecule of the amino acid selected from the five kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, and glycine is interposed between a self-assembled monolayer and a molecule of a protein. A method for immobilizing an protein on a self-assembled monolayer includes the following steps (a) and (b) in this order: a step (a) of preparing a substrate including one molecule of an amino acid and the self-assembled monolayer and a step (b) of supplying the protein to the substrate to form a peptide bond represented by a predetermined chemical formula as a result of reaction between the carboxyl group of the one molecular of the amino acid and the amino group of the protein.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This is a continuation of International application No. PCT/JP2011/007239, with international filling date of Dec. 22, 2011, which claims priority of Japanese Patent Application No. 2011-151573, filed on Jul. 8, 2011, the contents of all of which are hereby incorporated by reference. 
         [0002]    Further, it is noted that International Patent Publication Nos. WO2011/089903, WO2012/029202, WO2012/053138, WO2012/168988 and WO2013/005269 are commonly owned by the Assignee of the present application. 
     
    
     BACKGROUND 
       [0003]    The present disclosure relates to a method for immobilizing a protein on a self-assembled monolayer. 
         [0004]    A biosensor is used to detect or quantify a target substance contained in a sample. Some biosensors include protein capable of binding to the target substance to detect or quantify the target substance. More particularly, a biosensor for detecting or quantifying an antigen includes an antibody capable for binding specifically to the antigen. Similarly, biosensors for detecting or quantifying biotin and glucose include streptavidin and glucose oxidase, respectively. 
         [0005]    When a sample containing the target substance is supplied to the biosensor including protein capable of binding to the target substance, the target substance is bound to the protein to detect or quantify the target substance. 
         [0006]    International Patent Publication No. WO00/04382 discloses a conventional biosensor including protein. WO00/04382 corresponds to Japanese Publication of a translation of PCT international application No. 2002-520618 (see, e.g. Page 24, lines 23-26, Page 25, lines 3-20, Page 25, line 27-Page 26, line 13, Page 26, lines 14-22, Page 28, lines 21-23, Page 32, lines 3-29, Page 35, and line 21-Page 36, line 21 of WO00/04382 or paragraphs [0080], [0082], [0084], [0085], [0095], [0109], [0118], and [0119] of the corresponding Japanese Publication).  FIG. 2  shows a biosensor disclosed in FIG. 7 of Patent Literature 1. 
         [0007]    According to the description regarding FIG. 7 of WO00/04382, the biosensor is used for screening an activity of a biomolecule. The biosensor includes a monolayer  7 , an affinity tag 8, an adaptor molecule 9, and a protein  10 . The monolayer  7  is composed of a self-assembled monolayer represented by chemical formula: X—R—Y (see, Page 24, lines 23-26, Page 25, lines 3-20, Page 25, line 27-Page 26, line 13, and Page 26, lines 14-22 of WO00/04382; or paragraphs [0080], [0082], [0084] and [0085] of the corresponding Japanese Publication). Examples of X, R, and Y are HS—, an alkane, and a carboxyl group, respectively (see, Page 25, lines 3-20, Page 25, lines 27-Page 26, line 13, and Page 28, lines 21-23 of WO00/04382; or paragraphs [0084], [0085], and [0095] of the corresponding Japanese Publication). 
       BRIEF SUMMARY 
     Technical Problem 
       [0008]    In order to improve the detection sensitivity or the quantification accuracy of the target substance, it is required to increase an amount of protein to be immobilized on the biosensor. 
         [0009]    The present inventor has discovered that the amount of the immobilized protein per unit area was increased significantly by binding one molecule amino acid selected from the group consisting of cysteine, lysine, histidine, phenylalanine, and glycine to a self-assembled monolayer and then immobilizing protein. The present subject matter has been provided on the basis of the discovery. 
         [0010]    The purpose of the present disclosure is to provide a method for increasing an amount of protein to be immobilized on the self-assembled monolayer, and a sensor with the protein immobilized in accordance with the same method. 
       Solution to Problem 
       [0011]    A method for immobilizing a protein on a self-assembled monolayer includes the following steps. Step (a) is a step of preparing a substrate including one molecule of an amino acid and the self-assembled monolayer. The one molecule of the amino acid is bound to the self-assembled monolayer through a peptide bond represented by the following chemical formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of one molecule of the amino acid. The one molecular of the amino acid is selected from the five kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, and glycine. Step (b) is a step of supplying the protein to the substrate to form a peptide bond represented by the following chemical formula (II) as a result of reaction between the carboxyl group of the one molecule of the amino acid and the amino group of the protein: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. 
         [0012]    In one embodiment, the step (a) may include the following steps (a1) and (a2). Step (a1) is a step of preparing the substrate comprising the self-assembled monolayer on the surface thereof, the self-assembled monolayer having a carboxyl acid at one end. Step (a2) is a step of supplying the one molecule of the amino acid to form the peptide bond represented by the chemical formula (I) as a result of reaction between the carboxyl group of the one end of the self-assembled monolayer and the amino group of the one molecule of the amino acid. 
         [0013]    In one embodiment, the method may further include, between the step (a) and the step (b), a step (ab) of activating the carboxyl group of the one molecule of the amino acid with a mixture of N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. 
         [0014]    In one embodiment, the method may further includes, between the step (a1) and the step (a2), a step (a1a) of activating the carboxyl group of the self-assembled monolayer with a mixture of N-Hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. 
         [0015]    In one embodiment, the chemical formula (II) may be represented by the following chemical formula (III): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. 
         [0016]    One aspect of the present disclosure is a sensor including a self-assembled monolayer, one molecule of an amino acid, and a protein. The one molecule of the amino acid is interposed between the self-assembled monolayer and the protein, and the protein is bound to the self-assembled monolayer through two peptide bonds represented by the following chemical formula (II): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. The one molecule of the amino acid is selected from the five kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, and glycine. 
         [0017]    In one embodiment, the chemical formula (II) may be represented by the following chemical formula (III): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. 
         [0018]    One aspect of the present disclosure is a method for detecting or quantifying a target substance contained in a sample with use of a sensor. The method includes the following steps. Step (a) is a step of preparing the sensor including a self-assembled monolayer, one molecule of an amino acid, and a protein. The one molecule of the amino acid is interposed between the self-assembled monolayer and the protein, and the protein is bound to the self-assembled monolayer through two peptide bonds represented by the following chemical formula (II): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. The one molecule of the amino acid is selected from the five kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, and glycine. Step (b) is a step of supplying the sample to the sensor to bind the target substance to the protein. Step (c) is a step of detecting the target substance bound in the step (b), or quantifying the target substance contained in the sample from the amount of the target substance bound in the step (b). 
         [0019]    In one embodiment, the chemical formula (II) may be represented by the following chemical formula (III): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. 
       Advantageous Effect 
       [0020]    The present subject matter can increase significantly the amount of the protein to be immobilized per unit area. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1  shows an exemplary schematic view of a method according to one embodiment of the present disclosure. 
           [0022]      FIG. 2  corresponds to FIG. 7 of WO00/04382. 
           [0023]      FIG. 3  shows a schematic view of a method according to the prior art. 
       
    
    
     DETAILED DESCRIPTION 
       [0024]    The embodiment of the present disclosure is described below with reference to  FIG. 1 . 
         [0025]      FIG. 1  shows an exemplary method according to the present disclosure for immobilizing protein on a self-assembled monolayer. 
         [0026]    Preferably, a substrate  1  is a gold substrate. An example of the gold substrate is a substrate having gold uniformly on its surface. Specifically, the gold substrate may be a substrate having a gold film formed by a sputtering method on the surface of glass, plastic, or SiO 2 . 
         [0027]    First, the substrate  1  is immersed into a solvent containing an alkanethiol. Preferably, the substrate is washed before immersed. The alkanethiol has a carboxyl group at the end thereof. It is preferable that the alkanethiol has the carbon number within the range from six to eighteen. Thus, a self-assembled monolayer  2  is formed on the substrate  1 . 
         [0028]    The preferred concentration of the alkanethiol is approximately 1 mM to 10 mM. The solvent is not limited to, as long as it dissolves the alkanethiol. An example of the preferred solvent is ethanol, dimethyl sulfoxide (hereinafter, referred to as “DMSO”), and dioxane. The preferred immersing period is approximately 12 to 48 hours. 
         [0029]    Next, an amino acid  3  is supplied to the self-assembled monolayer  2 . The carboxyl group (—COOH), which is located at the top end of the self-assembled monolayer  2 , reacts with an amino group (—NH 2 ) of the amino acid  3  to form a peptide bond represented by the following the chemical formula (I): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. 
         [0030]    In the chemical formula (I), one molecule of the amino acid  3  binds to the self-assembled monolayer  2 . 
         [0031]    The amino acid  3  is selected from five kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine, and glycine. In other words, in the chemical formula (I), R is the side chain of these five kinds of amino acids. 
         [0032]    When the amino acid  3  is supplied to the self-assembled monolayer  2 , two or more kinds of amino acids may be supplied simultaneously. In other words, when a solution containing the amino acid  3  is supplied to the self-assembled monolayer  2 , the solution may contain two or more kinds of the amino acids  3 . In light of uniform bind of the protein to the amino acid  3 , which is described later, it is preferred that the solution contains a sole kind of amino acid. 
         [0033]    Subsequently, protein  4  is supplied. The 5′-terminal amino group of the protein  4  reacts with the carboxyl group of the amino acid  3 . The amino group of the lysine included in the protein also reacts with the carboxyl group of the amino acid  3 . Thus, two peptide bonds represented by the following chemical formula (II) are formed to obtain a sensor: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. 
         [0034]    One molecule of the protein  4  has only one N-terminus (the start of the protein terminated by an amino acid with a free amine group), corresponding to the 5′ end of mRNA encoding the protein, whereas the one molecule of the protein  4  has a lot of lysine groups having a free amine group. Therefore, almost all of the chemical formula (II) is represented more specifically by the following chemical formula (III): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R represents the side chain of the one molecule of the amino acid. 
         [0035]    The obtained sensor is used for detecting or quantifying the target substance contained in the sample. 
       EXAMPLES 
       [0036]    The following examples and comparative examples describe the present subject matter in more detail. 
       Comparative Example A 1 
       [0037]    As shown in  FIG. 3 , Protein A was bound directly with an amide coupling reaction to a carboxyl group located at the top end of self-assembled alkanethiol formed on the gold surface to immobilize the Protein A. The procedure and the results were described below. It is well-known that Protein A is a protein which constitutes five percent of the cell wall of  staphylococcus aureus  and is abbreviated as “SpA”. 
       [Preparation of a Sample Solution] 
       [0038]    A sample solution of 16-Mercaptohexadecanoic acid with final concentration of 10 mM was prepared. The solvent thereof was ethanol. 
       [Formation of a Self-Assembled Monolayer] 
       [0039]    A gold substrate (available from GE healthcare company, BR-1004-05) with gold vapor-deposited on glass was used as a substrate  1 . The substrate  1  was washed for ten minutes with a piranha solution containing concentrated sulfuric acid and 30% hydrogen peroxide water. The volume ratio of the concentrated sulfuric acid to the 30% hydrogen peroxide water contained in the piranha solution was 3:1. 
         [0040]    Subsequently, the gold substrate was immersed in the sample solution for 18 hours to form a self-assembled monolayer on the surface of the gold substrate. Finally, the substrate  1  was washed with pure water and dried. 
       [Immobilization of Protein] 
       [0041]    As protein, Protein A was bound to the carboxyl acid group located at the top end of the 16-Mercaptohexadecanoic acid which formed the self-assembled monolayer to immobilize the Protein A. 
         [0042]    Specifically, the carboxyl acid group located at the top end of the 16-Mercaptohexadecanoic acid was activated with use of 35 microliters of a mixture of 0.1M NHS (N-Hydroxysuccinimide) and 0.4M EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). Subsequently, 35 microliters of the Protein A (40 ug/ml) was added at the flow rate of 5 microliters/minute. Thus, the carboxyl acid of the 16-Mercaptohexadecanoic acid was coupled with the amino group of the Protein A. 
       Example A1 
       [0043]    Experiment was conducted similarly to the comparative example A1 except that glycine was supplied as the one molecule of the amino acid between the formation of the self-assembled monolayer and the immobilization of the Protein A. The procedure and the results are described below. 
       [Immobilization of Amino Acid (Glycine)] 
       [0044]    Glycine was bound with the carboxyl group located at the top end of the 16-Mercaptohexadecanoic acid which formed the self-assembled monolayer  2  to immobilize the glycine. 
         [0045]    Specifically, after the carboxyl group was activated similarly to the comparative example A1, 35 microliters of 0.1M glycine (pH: 8.9) was added at the flow rate of 5 microliters/minute. Thus, the carboxyl group of 16-Mercaptohexadecanoic acid was coupled with the amino group of the glycine. 
       [Immobilization of Protein] 
       [0046]    Subsequently, Protein A was bound to the carboxyl group of the glycine to immobilize the Protein A. Specifically, after the carboxyl group of the glycine was activated similarly to the above, 35 microliters of Protein A (concentration: 250 micrograms/ml) was added at the flow rate of 5 microliters/minute. Thus, the carboxyl group was coupled with the 5′-terminal amino acid of the Protein A or the amino group of the lysine included in the Protein A. 
       [Comparison of the Immobilization Amounts] 
       [0047]    The immobilization amounts in the example A1 and in the comparative example A1 were measured with use of an SPR device, Biacore 3000 (available from GE healthcare company). 
         [0048]    The term “immobilization amount” means the amount of the protein immobilized per unit area. 
       Comparative Examples A2-A16 
       [0049]    Serine, alanine, glutaminic acid, methionine, leucine, valine, threonine, isoleucine, tyrosine, asparagine, tryptophan, aspartic acid, arginine, proline and glutamine were used instead of glycine, and each immobilization amount was measured similarly to the case of the example A1. 
       Examples A2-A5 
       [0050]    Cysteine, lysine, histidine and phenylalanine were used instead of glycine, and each immobilization amount was measured similarly to the case of the example A1. 
         [0051]    Table 1 shows the immobilization amounts of Protein A in accordance with the examples A1-A5 and the comparative examples A1-A16. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
             
             
               
                   
                 Example A2 
                 Cysteine 
                 17.96117 
               
               
                   
                 Example A3 
                 Lysine 
                 14.27184 
               
               
                   
                 Example A4 
                 Histidine 
                 11.35922 
               
               
                   
                 Example A5 
                 Phenylalanine 
                 10.87379 
               
               
                   
                 Example A1 
                 Glycine 
                 9.708738 
               
               
                   
                 Comparative Example A16 
                 Asparagine 
                 9.223301 
               
               
                   
                 Comparative Example A2 
                 Methionine 
                 9.126214 
               
               
                   
                 Comparative Example A3 
                 Serine 
                 8.932039 
               
               
                   
                 Comparative Example A4 
                 Tyrosine 
                 6.850394 
               
               
                   
                 Comparative Example A5 
                 Tryptophan 
                 8.349515 
               
               
                   
                 Comparative Example A6 
                 Leucine 
                 7.76699 
               
               
                   
                 Comparative Example A7 
                 Glutamine 
                 7.378641 
               
               
                   
                 Comparative Example A8 
                 Alanine 
                 7.281553 
               
               
                   
                 Comparative Example A9 
                 Isoleucine 
                 5.533981 
               
               
                   
                 Comparative Example A10 
                 Threonine 
                 5.242718 
               
               
                   
                 Comparative Example A11 
                 Proline 
                 4.07767 
               
               
                   
                 Comparative Example A12 
                 Glutamic acid 
                 3.203883 
               
               
                   
                 Comparative Example A13 
                 Aspartic acid 
                 2.427184 
               
               
                   
                 Comparative Example A14 
                 Valine 
                 2.106796 
               
               
                   
                 Comparative Example A15 
                 Argnine 
                 0.621359 
               
               
                   
                 Comparative Example A1 
                 (None) 
                 1 
               
               
                   
                   
               
             
          
         
       
     
       Examples B1-B5 and Comparative examples B1-B16 
       [0052]    Experiments similar to the example A1-A5 and the comparative examples A1-A16 were conducted except that streptavidin was used instead of Protein A. 
         [0053]    Table 2 shows the immobilization amounts of the streptavidin in accordance with the examples B1-B5 and the comparative examples B1-B16. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
             
             
               
                   
                 Example B2 
                 Lysine 
                 33 
               
               
                   
                 Example B3 
                 Histidine 
                 32.2 
               
               
                   
                 Example B4 
                 Phenylalanine 
                 28.8 
               
               
                   
                 Example B5 
                 Cysteine 
                 26.9 
               
               
                   
                 Example B1 
                 Glycine 
                 25.6 
               
               
                   
                 Comparative Example B16 
                 Methionine 
                 25.6 
               
               
                   
                 Comparative Example B2 
                 Glutamic acid 
                 24.2 
               
               
                   
                 Comparative Example B3 
                 Tyrosine 
                 24.1 
               
               
                   
                 Comparative Example B4 
                 Alanine 
                 21.8 
               
               
                   
                 Comparative Example B5 
                 Serine 
                 20.5 
               
               
                   
                 Comparative Example B6 
                 Aspartic acid 
                 19.7 
               
               
                   
                 Comparative Example B7 
                 Asparagine 
                 18.6 
               
               
                   
                 Comparative Example B8 
                 Leucine 
                 12.9 
               
               
                   
                 Comparative Example B9 
                 Tryptophan 
                 12 
               
               
                   
                 Comparative Example B10 
                 Threonine 
                 9.1 
               
               
                   
                 Comparative Example B11 
                 Isoleucine 
                 6.4 
               
               
                   
                 Comparative Example B12 
                 Valine 
                 6.1 
               
               
                   
                 Comparative Example B13 
                 Glutamine 
                 3.6 
               
               
                   
                 Comparative Example B14 
                 Proline 
                 3.1 
               
               
                   
                 Comparative Example B15 
                 Argnine 
                 2.5 
               
               
                   
                 Comparative Example B1 
                 (None) 
                 1 
               
               
                   
                   
               
             
          
         
       
     
       Examples C1-C5 and Comparative examples C1-C16 
       [0054]    Experiments similar to the example A1-A5 and the comparative examples A1-A16 were conducted except that glucose oxidase was used instead of Protein A. 
         [0055]    Table 3 shows the immobilization amounts of the glucose oxidase in accordance with the examples C1-C5 and the comparative examples C1-C16. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 3 
               
               
                   
                   
               
             
             
               
                   
                 Example C2 
                 Cysteine 
                 37.69685 
               
               
                   
                 Example C3 
                 Lysine 
                 36.59207 
               
               
                   
                 Example C4 
                 Histidine 
                 36.16066 
               
               
                   
                 Example C5 
                 Phenylalanine 
                 30.35305 
               
               
                   
                 Example C1 
                 Glycine 
                 30.32874 
               
               
                   
                 Comparative Example C16 
                 Methionine 
                 29.62198 
               
               
                   
                 Comparative Example C2 
                 Serine 
                 29.40409 
               
               
                   
                 Comparative Example C3 
                 Alanine 
                 26.89383 
               
               
                   
                 Comparative Example C4 
                 Asparagine 
                 25.171 
               
               
                   
                 Comparative Example C5 
                 Leucine 
                 23.02633 
               
               
                   
                 Comparative Example C6 
                 Tyrosine 
                 22.1215 
               
               
                   
                 Comparative Example C7 
                 Glutamic acid 
                 20.36339 
               
               
                   
                 Comparative Example C8 
                 Isoleucine 
                 17.82311 
               
               
                   
                 Comparative Example C9 
                 Threonine 
                 15.35175 
               
               
                   
                 Comparative Example C10 
                 Aspartic acid 
                 14.48565 
               
               
                   
                 Comparative Example C11 
                 Tryptophan 
                 12.91537 
               
               
                   
                 Comparative Example C12 
                 Valine 
                 10.40278 
               
               
                   
                 Comparative Example C13 
                 Argnine 
                 6.055117 
               
               
                   
                 Comparative Example C14 
                 Proline 
                 5.792629 
               
               
                   
                 Comparative Example C15 
                 Glutamine 
                 1.202646 
               
               
                   
                 Comparative Example C1 
                 (None) 
                 1 
               
               
                   
                   
               
             
          
         
       
     
       Examples D1-D5 and Comparative Examples D1-D16 
       [0056]    Experiments similar to the example A1-A5 and the comparative examples A1-A16 were conducted except that antibody was used instead of Protein A. 
         [0057]    Table 4 shows the immobilization amounts of the antibody in accordance with the examples D1-D5 and the comparative examples D1-D16. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
             
             
               
                   
                 Example D2 
                 Histidine 
                 23.86045 
               
               
                   
                 Example D3 
                 Cysteine 
                 22.74856 
               
               
                   
                 Example D4 
                 Lysine 
                 20.91865 
               
               
                   
                 Example D5 
                 Phenylalanine 
                 18.86891 
               
               
                   
                 Example D1 
                 Glycine 
                 18.63296 
               
               
                   
                 Comparative Example D16 
                 Tryptophan 
                 17.46708 
               
               
                   
                 Comparative Example D2 
                 Methionine 
                 16.50562 
               
               
                   
                 Comparative Example D3 
                 Serine 
                 16.01948 
               
               
                   
                 Comparative Example D4 
                 Asparagine 
                 15.96672 
               
               
                   
                 Comparative Example D5 
                 Tyrosine 
                 15.85254 
               
               
                   
                 Comparative Example D6 
                 Alanine 
                 15.40134 
               
               
                   
                 Comparative Example D7 
                 Glutamic acid 
                 14.41335 
               
               
                   
                 Comparative Example D8 
                 Threonine 
                 13.00732 
               
               
                   
                 Comparative Example D9 
                 Leucine 
                 8.816629 
               
               
                   
                 Comparative Example D10 
                 Valine 
                 5.974514 
               
               
                   
                 Comparative Example D11 
                 Isoleucine 
                 5.701262 
               
               
                   
                 Comparative Example D12 
                 Aspartic acid 
                 3.676188 
               
               
                   
                 Comparative Example D13 
                 Proline 
                 3.276342 
               
               
                   
                 Comparative Example D14 
                 Argnine 
                 2.457678 
               
               
                   
                 Comparative Example D15 
                 Glutamine 
                 1.171725 
               
               
                   
                 Comparative Example D1 
                 (None) 
                 1 
               
               
                   
                   
               
             
          
         
       
     
       Examples E1-E5 and Comparative examples E1-E16 
       [0058]    Experiments similar to the example A1-A5 and the comparative examples A1-A16 were conducted except that albumin was used instead of Protein A. 
         [0059]    Table 5 shows the immobilization amounts of the antibody in accordance with the examples E1-E5 and the comparative examples E1-E16. 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
             
             
               
                   
                 Example E2 
                 Cysteine 
                 19.49204 
               
               
                   
                 Example E3 
                 Lysine 
                 18.39829 
               
               
                   
                 Example E4 
                 Histidine 
                 16.81413 
               
               
                   
                 Example E5 
                 Phenylalanine 
                 15.16347 
               
               
                   
                 Example E1 
                 Glycine 
                 14.39286 
               
               
                   
                 Comparative Example E16 
                 Serine 
                 12.94221 
               
               
                   
                 Comparative Example E2 
                 Alanine 
                 12.7583 
               
               
                   
                 Comparative Example E3 
                 Glutamic acid 
                 11.42908 
               
               
                   
                 Comparative Example E4 
                 Methionine 
                 11.05119 
               
               
                   
                 Comparative Example E5 
                 Leucine 
                 10.66873 
               
               
                   
                 Comparative Example E6 
                 Valine 
                 8.958131 
               
               
                   
                 Comparative Example E7 
                 Threonine 
                 8.8923 
               
               
                   
                 Comparative Example E8 
                 Isoleucine 
                 8.802846 
               
               
                   
                 Comparative Example E9 
                 Tyrosine 
                 8.288947 
               
               
                   
                 Comparative Example E10 
                 Asparagine 
                 8.018876 
               
               
                   
                 Comparative Example E11 
                 Tryptophan 
                 7.88124 
               
               
                   
                 Comparative Example E12 
                 Aspartic acid 
                 6.962646 
               
               
                   
                 Comparative Example E13 
                 Argnine 
                 5.856666 
               
               
                   
                 Comparative Example E14 
                 Proline 
                 3.829463 
               
               
                   
                 Comparative Example E15 
                 Glutamine 
                 3.654396 
               
               
                   
                 Comparative Example E1 
                 (None) 
                 1 
               
               
                   
                   
               
             
          
         
       
     
         [0060]    A skilled person would understand the followings from Table 1 to Table 5. 
         [0061]    When the one molecule of the amino acid selected from the five kinds of amino acids consisting of cysteine, lysine, histidine, phenylalanine and glycine is interposed between the self-assembled monolayer and the protein, the immobilization amount of the protein per unit area is increased, compared to the case where the one molecule of the amino acid selected from other fifteen kinds of the amino acid is used or to the case where one molecule of the amino acid is not used. 
       INDUSTRIAL APPLICABILITY 
       [0062]    The present subject matter can increase significantly the amount of the protein to be immobilized per unit area. This improves the sensitivity or the accuracy of the biosensor. The biosensor may be used for an inspection or a diagnosis which requires the detection or the quantification of the target substance contained in the living sample derived from a patient at a clinical practice. 
         [0063]    In the present patent application, Protein A, streptavidin, glucose oxidase, antibody and albumin may be excluded from the term “protein” used in the claims. 
       REFERENTIAL SIGNS LIST 
       [0000]    
       
           1 : Gold substrate 
           2 : Alkanethiol 
           3 : Amino Acid 
           4 : Protein