Patent Publication Number: US-2010112578-A1

Title: Test chip, detection apparatus, and method for detecting analyte

Description:
RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. §119 to Japanese Patent Application No. 2008-281584 filed on Oct. 31, 2008, and Japanese Patent Application No. 2009-226321 filed on Sep. 30, 2009, the entire content of which is hereby incorporated by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to a test chip, a detection apparatus and a method for detecting an analyte. 
     BACKGROUND 
     In clinical examination and diagnosis of diseases, disease-derived genes, proteins and the like contained in biological samples are detected by gene detection methods and immunological detection methods. Specific examples include immunochromatography, latex agglutination, enzyme immunoassay, chemiluminescent immunoassay, gene amplification PCR, and the like. 
     In these detection methods, however, there is room for improvement from the viewpoint of simplicity, rapidity, and cost. 
     Consequently, EP1947452 has proposed a method wherein an electric current generated from a sensitizing dye by photoexcitation is utilized in detecting an analyte. In this method, a semiconductor layer is first formed on an electrode, and a probe capable of binding to the analyte is immobilized on the semiconductor layer. Then, the analyte modified with a sensitizing dye is trapped with the probe substance, and then the sensitizing dye with which the analyte is modified is irradiated with a light for exciting the sensitizing dye. As a result, electrons are emitted from the sensitizing dye with which the analyte is modified, and when the emitted electrons are received by the semiconductor layer, an electric current is generated and detected. By using a crosslinking agent such as a silane coupling agent, the probe has been immobilized on the semiconductor layer. However, the silane coupling agent has low conductivity to reduce the efficiency of detection of the electric current, and thus has a problem of low sensitivity in detection of the analyte. 
     SUMMARY OF THE INVENTION 
     The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary. 
     A first aspect of the present invention is a test chip for detecting an analyte modified with a modulator releasing electrons upon photoexcitation, comprising: a semiconductor electrode part including a metal layer formed on a semiconductor layer; a probe immobilized on the metal layer, the probe trapping the analyte; and a counter electrode part including a conductive layer. 
     A second aspect of the present invention is an apparatus for detecting an analyte modified with a modulator releasing electrons upon photoexcitation, comprising: a test chip receiving part capable of receiving a test chip, a light source for irradiating a modulator with light to photoexcite the modulator modifying the analyte; and an electric current measuring part for measuring an electric current flowing generated from the photoexcited modulator modifying the analyte, wherein the test chip comprises: a semiconductor electrode part including a metal layer formed on a semiconductor layer; a probe immobilized on a metal layer, the probe trapping an analyte; and a counter electrode part including a conductor layer. 
     A third aspect of the present invention is a method for detecting an analyte modified with a modulator releasing electrons upon photoexcitation, comprising: trapping an analyte in a sample by using a test chip, wherein the test chip comprises: a semiconductor electrode part including a metal layer formed on a semiconductor layer; a probe immobilized on the metal layer, the probe trapping an analyte; and a counter electrode part of a conductive layer: modifying the analyte with a modulator; irradiating the modulator with light to photoexcite the modulator modifying the analyte; and detecting an electric current flowing generated from the photoexcited modulator. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view showing a detection apparatus  1  in accordance with one embodiment of the present invention; 
         FIG. 2  is a block diagram showing the constitution of the detection apparatus  1 ; 
         FIG. 3  is a perspective view showing a test chip  4  used in the detection apparatus  1 ; 
         FIG. 4  is a perspective view showing an upper plate having a semiconductor electrode part  15  of the test chip  4 ; 
         FIG. 5  is a perspective view showing a lower plate having a counter electrode part  16  of the test chip  4 ; 
         FIG. 6  is a perspective view of the test chip  4  from which the upper substrate  13  was detached; 
         FIG. 7  is a sectional view showing the constitution of the test chip  4 ; 
         FIG. 8  is a schematic view showing the constitution of the semiconductor electrode part  15  and a counter electrode part  18  in the test chip  4 ; 
         FIG. 9  is a flowchart showing a method of injecting an analyte to the test chip  4  by the user; 
         FIG. 10  is a flowchart showing the procedure of detection operation of the detection apparatus  1 ; 
         FIG. 11  is a schematic view of the semiconductor electrode part  15  at the time of hybridization and at the time of addition of an electrolytic solution; 
         FIG. 12  is a graph showing photocurrent values obtained by measurement in Example 1 and Comparative Example 1; 
         FIG. 13  is a graph of photocurrent values detected in Example 2 and Comparative Example 2; 
         FIG. 14  is a graph showing a modulator-derived electric current value among data obtained in Example 2 and Comparative Example 2; 
         FIG. 15  is a graph of photocurrent values detected in Example 3, Comparative Example 3 and Comparative Example 4; 
         FIG. 16  is a graph of photocurrent values detected in Example 4, Comparative Example 5 and Comparative Example 6; and 
         FIG. 17  is a graph of S/N ratio in each film thickness detected in Example 5. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Hereinafter, the detection chip, the detection apparatus and the method for detecting an analyte according to the present invention will be described with reference to the drawings. 
     (Constitution of the Detection Apparatus) 
       FIG. 1  is a perspective view of the detection apparatus in accordance with one embodiment of the present invention. The detection apparatus is a apparatus for detecting an analyte having specific binding property, such as a nucleic acid, a protein or a peptide which was collected from living cells or synthesized artificially. The detection apparatus  1  can for example detect, in an analyte sample, mRNA of human papillomavirus (referred to hereinafter as HPV) that is a causative virus for cervical cancer. 
     The detection apparatus  1  in this embodiment includes a chip receiving part  3  into which a test chip  4  is inserted and a display  2  on which a detection result is displayed. The test chip  4  includes a sample injection port  11 . 
     The test chip  4  is a disposable HPV detection chip and is inserted into the chip receiving part  3  of the detection apparatus  1 . The test chip  4  has a function of trapping mRNA of HPV modified with a modulator generating electrons by photoexcitation upon injecting an analyte sample through the sample injection port  11 . 
       FIG. 2  is a block diagram showing the constitution of the detection apparatus  1 . The detection apparatus  1  includes a light source  5 , an ammeter  6 , a power supply  32 , an A/D converter  7 , a controller  8  and a display  2 . 
     The light source  5  applies a light to a modulator that modifies mRNA of HPV trapped by the test chip  4 , thereby exciting the modulator. The ammeter  6  measures an electric current flowing due to electrons emitted from the excited modulator. The power supply  32  applies a predetermined potential to an electrode arranged in the test chip  4 . The A/D converter  7  converts an electric current value measured by the ammeter  6  into a digital value. The controller  8  is composed of CPU, ROM and RAM, and regulates the operation of the light source  5 , the ammeter  6 , and the display  2 . On the basis of a previously prepared calibration curve showing the relationship between electric current values and the amounts of HPV, the controller  8  makes a rough estimate of the amount of HPV in an analyte sample, from the digital value into which the electric current value was converted by the A/D converter  7 . The display  2  displays that amount of HPV in an analyte sample which was estimated roughly by the controller  8 . 
     (Constitution of the Test Chip  4 ) 
     The constitution of the test chip  4  used in the detection apparatus  1  will be described with reference to  FIGS. 3 to 8 . 
       FIG. 3  is a perspective view of the test chip  4 . The test chip  4  includes a lower substrate  16 , an upper substrate  13  arranged above the lower substrate  16 , and a silicon rubber  12  put between the lower substrate  16  and the upper substrate  13 . The upper substrate  13  is provided with the sample injection port  11  communicating therein. 
       FIG. 4  is a perspective view of the upper substrate  13  after the test chip  4  in  FIG. 3  is rotated horizontally right by 90° and then rotated vertically by 180°. The semiconductor electrode part  15 , and an electrode lead  14  connected to the semiconductor electrode part  15 , are formed on the surface of the upper substrate  13 . The upper substrate  13  is formed of silicon dioxide (SiO 2 ), and the electrode lead  14  is formed of 2 layers consisting of indium tin oxide (ITO) and antimony-doped tin oxide (ATO). The semiconductor electrode part  15  will be described later with reference to  FIG. 8 . 
       FIG. 5  is a perspective view of the lower substrate  16  after the test chip  4  in  FIG. 3  is rotated horizontally right by 90°. A counter electrode part  18 , an electrode lead  17  connected to the counter electrode part  18 , a reference electrode  31 , and an electrode lead  30  connected to the reference electrode  31 , are formed on the surface of the lower substrate  16 . 
     The lower substrate  16  is formed of glass based on silicon dioxide (SiO 2 ), and the counter electrode part  18 , the electrode lead  17 , the reference electrode  31  and the electrode lead  30  are formed of platinum respectively. 
       FIG. 6  is a perspective view of the test chip  4  when the upper substrate  13  of the test chip  4  in  FIG. 3  is detached upwardly. The silicon rubber  12  as shown in  FIG. 6  is arranged so to surround the counter electrode part  18  and the reference electrode  31  on the lower substrate  16 . The electrode lead  17  connected to the counter electrode part  18  and the electrode lead  30  connected to the reference electrode  31  extend from the frame of the silicon rubber  12 . The electrode lead  17  and the electrode lead  30  extending from the frame are connected to the electric power  32 . 
     The sample injection port  11  arranged on the upper substrate  13  is a hole penetrating the upper substrate  13 . An analyte sample and an electrolytic solution described later are injected through the sample injection port  11  to the frame of the silicon rubber  12 . 
       FIG. 7  is a sectional view showing an A-A sectional constitution of the test chip  4  in  FIG. 3 . As shown in  FIG. 7 , the upper substrate  13  and lower substrate  16  contained in the test chip  4  are arranged via the silicon rubber  12 . A space  25  is formed between the upper substrate  13  and lower substrate  16 . The semiconductor electrode part  15  formed on the upper substrate  13  is opposed via the space  25  to the counter electrode part  18  and the reference electrode part  31  (not shown) formed on the lower substrate  16 . An analyte sample and an electrolytic solution described later are injected via the sample injection port  11  into the space  25 . 
     As shown in  FIG. 7 , the electrode lead  14  connected to the semiconductor electrode part  15  extends along the upper substrate  13  to the outside of the space  25 , and the electrode lead  17  connected to the counter electrode part  18  and the reference electrode  30  (not shown) connected to the reference electrode part  31  extend along the lower substrate  16  to the outside of the space  25 . The electrode lead  14  is connected to the ammeter  6 , and the electrode lead  17  and the electrode lead  30  are connected to the power supply  32 . 
     In this embodiment, the semiconductor electrode part  15  is formed on the surface of the upper substrate  13 , and the counter electrode part  18  and the reference electrode part  31  are formed on the surface of the lower substrate  16 , and the arrangement between the semiconductor electrode part  15 , the counter electrode part  18  and the reference electrode part  31  is not particularly limited as long as each electrode does not contact with other electrode and is arranged in the frame of the silicon rubber  12 . For example, the semiconductor electrode part  15 , the counter electrode part  18  and the reference electrode part  31  may be arranged on the same substrate. 
     The semiconductor electrode part  15  shown in  FIG. 4  will be further described.  FIG. 8  is a schematic diagram showing the constitution of the semiconductor electrode part  15  and counter electrode part  18 . 
     The semiconductor electrode part  15  includes a conductive layer  21  formed on the upper substrate  13 , a semiconductor layer  20  formed on the conductive layer  21 , and a metal layer  19  formed on the semiconductor layer  20 . The counter electrode part  18  is formed on the lower substrate  16 . 
     A probe  23  for trapping mRNA  24  of HPV modified with a modulator  22  generating electrons by photoexcitation is fixed on the metal layer  19  contained in the semiconductor electrode part  15 . The modulator  22  is a ruthenium complex and is bound, via a peptide bond, to the mRNA, thereby modifying the mRNA. 
     The electrode lead  14  connected to the semiconductor electrode part  15  is connected to the ammeter  6 , and the electrode lead  17  connected to the counter electrode part  18  and the electrode lead  30  connected to the reference electrode part  31  are connected to the power supply  32 . The ammeter  6  is connected to the power supply  32 , and an electric current flowing between the semiconductor electrode part  15  and the counter electrode part  18  is measured with the ammeter  6 . 
     The conductive layer  21  contained in the semiconductor electrode part  15  consists of 2 layers, that is, an indium tin oxide (ITO) layer formed by sputtering and antimony-doped tin oxide (ATO) formed by sputtering on the ITO layer. The semiconductor layer  20  consists of a titan oxide (TiO 2 ) layer formed by sputtering. The metal layer  19  consists of a gold (Au) layer formed by deposition. The counter electrode part  18  consists of a platinum layer formed by sputtering. 
     The probe  23  has a thiol group, and the probe  23  is immobilized on the metal layer  19  by binding a thiol group of the probe  23  to a gold atom of the metal layer  19 . This immobilization is carried out by dipping the metal layer  19  in an aqueous solution having the probe  23  dispersed therein. 
     (Detection Method Using HPV Detection Apparatus) 
     The method of using the detection apparatus  1  having the constitution described above is described with reference to  FIGS. 9 to 11 .  FIG. 9  is a flowchart showing the procedure of injecting an analyte into the detection chip  4  by the user.  FIG. 10  is a flowchart showing the procedure of detection operation of the detection apparatus  1 .  FIG. 11  is a schematic view of the semiconductor electrode part  15  at the time of hybridization and at the time of addition of an electrolytic solution. 
     From the flowchart in  FIG. 9 , the user in step S 1  injects an analyte sample into the sample injection port  11  of the sample chip  4 . This analyte sample is mRNA obtained by homogenization, extraction and purification from cervix cells. By step S 1 , the probe  23  on the metal layer  19  traps mRNA  24  of HPV in the analyte sample by hybridization, as shown in  FIG. 11 . 
     In step S 2 , the user discharges the solution in the test chip  4  from the sample injection port  11  and then washes the inside of the test chip  4  with a hybridization washing liquid. 
     In step S 3 , the user injects, through the sample injection port  11 , the modulator  22  containing a nucleotide sequence capable of binding to mRNA  24  of HPV. The injected modulator  22  modifies mRNA  24  trapped with the probe  23 . 
     In step S 4 , the user discharges the solution in the test chip  4  from the sample injection port  11  and washes the inside of the test chip  4  with a wash buffer. 
     In step S 5 , the user injects an electrolytic solution through the sample injection port  11 . This electrolyte is a mixture containing iodine as an electrolyte, tetrapropylammonium iodide as a supporting electrolyte, and an organic solvent consisting of acetonitrile and ethyl carbonate in a volume ratio of 6:4. When the electrolytic solution is added, the iodine contained in the electrolytic solution dissolves the metal layer  19 . 
     The dissolution of the metal layer  19  is described with reference to  FIG. 11 .  FIG. 11  is a schematic view of the semiconductor electrode part  15  at the time of hybridization and at the time of addition of an electrolytic solution. 
     The probe  23  is immobilized on the metal layer  19  by covalent bonding between a thiol group (SH group) of the probe  23  and a gold atom of the metal layer  19 . The covalent bonding is a strong bonding, so that in the hybridization step (step S 1 ) and in the washing step (step S 2 ), the probe  23  can be prevented from releasing from the metal layer  19 . 
     When the electrolytic solution is added, iodine contained in the electrolytic solution dissolves the metal layer  19  consisting of gold (Au), and the probe  23  is arranged on the semiconductor layer  20 . Electrons generated from the modulator  22  excited by light irradiation with the light source  5  are thereby fed efficiently to the semiconductor layer  20 . 
       FIG. 10  is a flowchart showing the detection procedure of the detection apparatus  1 . After the user performs the flow in  FIG. 9 , the user inserts the detection chip  4  into the chip insertion port  3  of the detection apparatus  1  shown in  FIG. 1 , and initiates measurement on the display  2 . 
     In step S 6 , electrode leads  14 ,  17  and  31  of the test chip  4  inserted into the detection apparatus  1  are connected to the ammeter  6  and power supply  32 . By the power supply  32 , a potential of 0 V relative to the reference electrode part  31  is applied to the semiconductor electrode part  15 . 
     In step S 7 , the light source  5  applies a laser light to the modulator  22  with which mRNA  24  of HPV is modified, thereby exciting the modulator  22 . The excited modulator  22  releases electrons, and the released electrons are transported into the semiconductor layer  20 . As a result, an electric current flows between the semiconductor electrode part  15  and the counter electrode part  18 . 
     In step S 8 , the electric current which due to electron movement in step S 5 , flows through the semiconductor electrode part  15  and the counter electrode part  18 , is measured with the ammeter  6 . The electric current value measured with the ammeter  6  is correlated with the number of modulators  22 , and thus HPV can be quantitatively determined on the basis of the measured electric current value. 
     In step S 9 , a digital value into which the electric current value was converted by the A/D converter  7  is inputted to the controller  8 . On the basis of a previously prepared calibration curve showing the relationship between electric current values and the amounts of HPV, the controller  8  makes a rough estimate of the amount of HPV in the analyte sample, from the digital value into which the electric current value was converted. To indicate on the display  2  the roughly estimated amount of HPV, a detection result screen is formed. 
     Then, in step S 10 , the detection result screen formed by the controller  8  is transmitted to, and displayed on, the display  2 . 
     In this embodiment, the analyte is mRNA  24  of HPV, but the analyte may be a nucleic acid, a protein or a peptide that is collected from living cells or artificially synthesized. The probe  23  may be a substance trapping an analyte, and may be for example a nucleic acid, a protein or a peptide. 
     Although the modulator  22  in this embodiment is a ruthenium complex, the modulator is not particularly limited as long as it is a substance to be excited by the light source  5 , thereby releasing electrons. Examples of such modulators include a metal complex, an organic dye and a quantum dot. Specific examples include metal phthalocyanine, a ruthenium complex, an osmium complex, an iron complex, a zinc complex, a 9-phenylxanthene dye, a cyanine dye, a metallocyanine dye, a xanthene dye, a triphenylmethane dye, an acridine dye, an oxazine dye, a coumarin dye, a merocyanine dye, a rhodacyanine dye, a polymethine dye, a porphyrin dye, a phthalocyanine dye, a rhodamine dye, a xanthene dye, a chlorophyll dye, an eosin dye, a mercurochrome dye, an indigo dye, and a cadmium selenide dye. 
     In this embodiment, the light source  5  is not particularly limited as long as it emits a light with wavelength that excites the substance with which an analyte is modified. Examples of such light sources include a laser, a light-emitting diode (LED), an inorganic electroluminescence element, an organic electroluminescence element, a white light source, and a white light source provided with an optical filter. 
     This embodiment is illustrated wherein mRNA  24  of HPV is trapped with the probe  23  and then the mRNA  24  of HPV is modified with the modulator  22 , but the mRNA  24  of HPV may be modified with the modulator  22  and then trapped with the probe  23 , whereby the mRNA  24  of HPV may be detected. When the analyte and the probe are nucleic acids, there is a method of intercalation wherein a modulator is bound to a double nucleic acid formed between an analyte and a probe for trapping the analyte. 
     Although the metal layer  19  in this embodiment is gold, the metal layer  19  may be any metal capable of binding to the probe  23 . Preferably, the metal layer  19  is a metal capable of covalently bonding to the probe  23 . More preferably, the metal layer  19  is a metal capable of binding to a thiol group of the probe  23 . For example, the metal layer  19  may be exemplified by gold, platinum, silver, palladium, nickel, mercury, rhodium, ruthenium, copper, or an alloy thereof. In this embodiment, the method of forming the metal layer  19  on the semiconductor layer  20  uses deposition, and may use sputtering, imprinting, screen printing, plating, or a sol-gel process. 
     In this embodiment, titanium oxide (TiO 2 ) is used as the semiconductor layer  20 , but the semiconductor layer  20  may be made of a substance capable of having energy levels at which it can receive electrons released from the modulator  22  upon excitation. Examples include semiconductors such as silicon and germanium and compound semiconductors or organic semiconductors such as titanium oxide (TiO 2 ), indium oxide (In 2 O 2 ), tin oxide (SnO 2 ), zinc oxide (ZnO), cadmium selenide (CdSe), cadmium sulfide (CdS), gallium nitride (GaN) and titanium nitride (TiN). 
     In this embodiment, the conductive layer  21  is formed of indium tin oxide (ITO) and antimony-doped tin oxide (ATO), but is not particularly limited as long as it is a conductive material. Examples include platinum, gold, silver and copper, and conductive ceramics and metal oxides. When the semiconductor layer  20  itself also functions as a conductive material, the conductive layer  21  can be omitted. 
     In this embodiment, the counter electrode part  18  is formed of platinum, but is not particularly limited as long as it is a conductive material. Examples include gold, silver and copper, and conductive ceramics and metal oxides. 
     In the embodiment described above, iodine is used as a substance for dissolving the metal layer  19  and as an electrolyte, but the substance for dissolving the metal layer  19  and the electrolyte may be different from each other. 
     In this embodiment, the probe  23  is bound directly to the metal layer  19 , but a crosslinking agent such as ethane dithiol may be present between the probe  23  and the metal layer  19 . 
     The detection apparatus  1  and the test chip  4  in this embodiment may be divided into a plurality of regions into which metal layers  19  are separated to immobilize the probe  23 , whereby the light irradiation with the light source  5  may be conducted individually for each region. A plurality of samples can thereby be measured with one semiconductor electrode part  15 . By immobilizing a plurality of probes on each region, many analytes and many measurement items can be measured with one test chip  4 . 
     By the power supply  32  in this embodiment, a potential of 0 V relative to the reference electrode part  31  is applied to the semiconductor electrode part  15 , but this reference electrode part  31  can be omitted. By the power supply  32  in this case, a potential of 0 V relative to the counter electrode part  18  can be applied to the semiconductor electrode part  15 . 
     EXAMPLES 
     Example 1 
     Examination of the Presence or Absence of Metal Layer on Semiconductor Electrode 
     (Preparation of Semiconductor Electrode Part) 
     Indium tin oxide (ITO) and antimony-doped tin oxide (ATO) were formed with a thickness of 100 nm as a conductive layer by sputtering on a substrate made of silicon dioxide (SiO 2 ). On this conductive layer, titanium oxide (TiO 2 ) was formed as a semiconductor layer of 10 nm in thickness by sputtering, and a gold thin film was formed as a metal layer of 10 nm in thickness thereon. By using a semiconductor layer containing titanium or chrome, the adhesion between the gold thin film and the semiconductor layer is improved. The member containing the conductive layer, the semiconductor layer and the metal layer is referred to the semiconductor electrode part. A semiconductor electrode lead for connection to an ammeter is connected to the semiconductor electrode part. 
     (Preparation of Counter Electrode Part) 
     A platinum thin layer formed with a thickness of 200 nm by sputtering on a substrate made of silicon dioxide (SiO 2 ) was used as the counter electrode part. To this counter electrode part was connected a counter lead for connection to an ammeter. 
     (Immobilization of Probe Substance) 
     A probe consisting of a DNA (24 bases) having a thiol group is immobilized on the metal layer of the semiconductor electrode part. First, the semiconductor electrode part was dipped for 18 hours in an aqueous solution having a nucleic acid (nucleic acid concentration 10 μM) dispersed therein. Thereafter, the semiconductor electrode part was washed with ultrapure water and dried for 30 minutes. As a result, the nucleic acid is immobilized on the metal layer by binding a thiol group of the nucleic acid to a gold atom of the metal layer. 
     (Preparation of Analyte) 
     As the analyte, a modulator-bound DNA having a nucleotide sequence complementary to the probe is prepared. As the modulator, a sensitizing dye Pulsar 650 (manufactured by Bio Search Technologies Japan) was used. This sensitizing dye is a ruthenium complex and is bound via a peptide bond to the DNA. 
     (Hybridization Between Analyte and Probe) 
     The analyte modified with the modulator is trapped with the probe on the semiconductor electrode part. 
     First, silicon rubber (thickness 0.2 mm) is arranged as a partition wall around the semiconductor electrode part. 10 μL of a hybridization solution is injected into the space formed by this silicon rubber. This hybridization solution is a mixture of the modulator-modified DNA (concentration 10 μM) and a hybridization buffer (Affymetrix). 
     Then, the silicon rubber was covered with a cover glass and subjected to hybridization such that the solution was not dried. Hybridization was carried out by leaving it at 45° C. for 1 hour. After hybridization, the sample was washed with a wash buffer A (Affymetrix) and ultrapure water, and then dried with a blower. 
     (Preparation of Electrolytic Solution) 
     A solvent consisting of acetonitrile (AN) and ethylene carbonate (EC) mixed in a volume ratio of 6:4 is prepared. As a supporting electrolyte salt, tetrapropylammonium iodide (NPr 4 I) is dissolved in an amount of 0.6 M. As an electrolyte, iodine is dissolved in an amount of 0.06 M. The resulting solution is used as the electrolytic solution. 
     (Measurement of Photocurrent) 
     Silicon rubber (thickness 0.2 mm) is arranged as a side wall around the substrate having the semiconductor electrode part where the analyte was hybridized with the probe. 10 μL of the electrolytic solution is injected into the space formed by this silicon rubber. Then, the semiconductor electrode part filled with the electrolytic solution is sealed from above with the substrate having the counter electrode part. The semiconductor electrode part and the counter electrode part are thereby contacted with the electrolytic solution. 
     Then, the semiconductor electrode lead and the counter electrode lead are connected to the ammeter. Then, the light source emits a light from the direction of the semiconductor electrode part to the counter electrode part. The light source is a laser light source having a wavelength of 473 nm and an intensity of 13 mW. As a result, the modulator is excited, electrons generated from the excited modulator are transported into the semiconductor layer, and an electric current flows through the semiconductor electrode part and the counter electrode part. This electric current value is measured. 
     Comparative Example 1 
     Comparative Example 1 is the same as in Example 1 except that the step of forming the metal layer on the semiconductor layer is not performed. 
     (Results) 
       FIG. 12  is a graph showing electric current values obtained in measurement of Example 1 and Comparative Example 1. By the method in Example 1, an electric current value of 229 nA was obtained. By the method in Comparative Example 1, on the other hand, an electric current value of 80 nA was obtained. 
     From this result, it could be seen that by forming the metal layer on the semiconductor layer, an electric current value as high as 3 times can be extracted, and the detection sensitivity of an electric current value was improved. 
     Example 2 
     Detection by Current Measurement of Analyte Modified with Modulator 
     (Preparation of Semiconductor Electrode Part) 
     Prepared in the same manner as in Example 1. 
     (Preparation of Counter Electrode) 
     Prepared in the same manner as in Example 1. 
     (Immobilization of Probe) 
     Performed in the same manner as in Example 1. 
     (Preparation of Analyte) 
     An analyte (analyte A) having a modulator bound to a DNA containing a nucleotide sequence complementary to the probe and an analyte (analyte B) having a modifier bound to a DNA not containing a nucleotide sequence complementary to the probe are prepared as analytes. 
     The modulator is a sensitizing dye Pulsar 650 (manufactured by Bio Search Technologies Japan). This sensitizing dye is a ruthenium complex and is bound to the DNA via a peptide bond. 
     (Trap of Analyte with Probe) 
     The analyte A or B is subjected to hybridization reaction with the probe on the metal layer. First, silicon rubber (thickness 0.2 mm) is arranged as a partition wall around the semiconductor electrode part. 10 μL of a hybridization solution is injected into the space formed by this silicon rubber. This hybridization solution is a mixture of the modulator-modified DNA (concentration 10 μM) and a hybridization buffer (Affymetrix). 
     Then, the silicon rubber was covered with a cover glass and subjected to hybridization such that the solution was not dried. Hybridization is carried out by leaving it at 45° C. for 1 hour. After hybridization, the sample was washed with a wash buffer A (Affymetrix) and ultrapure water, and then dried with a blower. 
     (Preparation of Electrolytic Solution) 
     Performed in the same manner as in Example 1. 
     (Measurement of Electric Current) 
     Silicon rubber (thickness 0.2 mm) is arranged as a side wall around the substrate having the semiconductor electrode part where the analyte A or B was hybridized with the probe. 10 μL of an electrolytic solution is injected into the space formed by this silicon rubber, and the semiconductor electrode part filled with the electrolytic solution is sealed from above with the substrate having the counter electrode part. The semiconductor electrode part and the counter electrode part are thereby contacted with the electrolytic solution. 
     Then, the semiconductor electrode lead and the counter electrode lead are connected to the ammeter, and the light source emits a light from the direction of the semiconductor electrode part to the counter electrode part. The light source was a laser light source having a wavelength of 473 nm and an intensity of 13 mW. The modulator with which the analyte is modified is thereby excited, electrons released from the excited modulator are transported into the semiconductor layer, and an electric current flows through the semiconductor electrode part and the counter electrode part. This electric current value was measured. 
     An electric current is measured as an electrode-derived electric current without conducting the step of hybridizing the analyte with the probe on the metal layer. The electrode-derived electric current refers to an electric current generated by irradiation of the electrode itself. 
     Comparative Example 2 
     The analyte was detected in the same manner as in Example 2 except that the step of forming the metal layer on the semiconductor layer was not carried out. 
     (Results) 
       FIG. 13  is a graph of electric current values detected in Example 2 and Comparative Example 2. 
     In Example 2, the electric current value detected was 36.9 nA when hybridization was conducted with the DNA (analyte A) having a nucleotide sequence complementary to the probe. 
     When hybridization was conducted with the DNA (analyte B) not having a nucleotide sequence complementary to the probe, the current value detected was 24.7 nA. This electric current value was equivalent to the electrode-derived electric current value of 24.9 nA. Accordingly, it can be confirmed that the electric current value detected by hybridization with the analyte A is not due to the unspecific adsorption of the analyte onto the semiconductor electrode part but due to specific detection by recognition of the sequence. 
       FIG. 14  is a graph showing modulator-derived electric current values among data obtained in Example 2 and Comparative Example 2. The modulator-derived electric current value refers to an electric current value obtained by subtracting an electrode-derived electric current value from an electric current value obtained by measuring the analyte. 
     The modulator-derived electric current value is greater by about 4.5 times in the metal layer-containing semiconductor electrode part (Example 2) than in the metal layer-free semiconductor electrode part (Comparative Example 2). 
     Example 3 
     Effect of the Semiconductor Electrode Using Modulator Excited with Long Wavelength 
     (Preparation of Semiconductor Electrode Part) 
     Indium tin oxide (ITO) was formed with a thickness of 100 nm as a conductive layer by sputtering on a substrate made of silicon dioxide (SiO 2 ). On this conductive layer, indium oxide (In 2 O 3 ) was formed as a semiconductor layer of 10 nm in thickness by sputtering, and a gold thin film was formed as a metal layer of 10 nm in thickness thereon. It was calcinated (150° C.) in an oxygen atmosphere, thereby improving the adhesion between the gold thin film and the semiconductor layer. The member containing the conductive layer, the semiconductor layer and the metal layer is referred to the semiconductor electrode part. A semiconductor electrode lead for connection to the ammeter is connected to the semiconductor electrode part. 
     (Preparation of Counter Electrode Part) 
     Prepared in the same manner as in Example 1. 
     (Immobilization of Probe Substance) 
     A probe consisting of a DNA (24 bases) having a thiol group is immobilized on the metal layer of the semiconductor electrode part. First, the semiconductor electrode part was dipped for 18 hours in an aqueous solution having a nucleic acid (nucleic acid concentration 10 μM) dispersed therein. Thereafter, the semiconductor electrode part was washed with ultrapure water and dried for 10 minutes. As a result, the nucleic acid is immobilized on the metal layer by binding a thiol group of the nucleic acid to a gold atom of the metal layer. 
     (Preparation of Analyte) 
     As the analyte, a modulator-bound DNA having a nucleotide sequence complementary to the probe is prepared. As the modulator, Alexa Fluor 750 (Invitrogen) was used. This modulator is an organic dye and is bound via a peptide bond to the DNA. 
     (Hybridization Between Analyte and Probe) 
     The dye-modified analyte is trapped with the probe on the semiconductor electrode part. 
     First, silicon rubber (thickness 0.2 mm) is arranged as a partition wall around the semiconductor electrode part. 10 μL of a hybridization solution is injected into the space formed by this silicon rubber. This hybridization solution was a mixture of the modulator-modified DNA (concentration 10 μM) and a hybridization buffer (Affymetrix). 
     Then, the silicon rubber was covered with a cover glass and subjected to hybridization such that the solution was not dried. Hybridization is carried out by leaving it at 45° C. for 1 hour. After hybridization, the sample was washed with a wash buffer A (Affymetrix) and ultrapure water, and then dried with a blower. 
     (Preparation of Electrolytic Solution) 
     First, a solvent consisting of acetonitrile (AN) and ethylene carbonate (EC) mixed in a volume ratio of 6:4 is prepared. As a supporting electrolyte salt, tetrapropylammonium iodide (NPr 4 I) is dissolved in an amount of 0.6 M. As an electrolyte, iodine is dissolved in an amount of 0.06 M. The resulting solution is used as the electrolytic solution. 
     (Measurement of Photocurrent) 
     Silicon rubber (thickness 0.2 mm) is arranged to surround the substrate having the semiconductor electrode where the analyte was hybridized with the probe. 10 μL of the electrolytic solution is injected into the space formed by this silicon rubber. The semiconductor electrode part filled with the electrolytic solution is sealed from above with the substrate having the counter electrode part. The semiconductor electrode part, the counter electrode part and the reference electrode part are thereby contacted with the electrolytic solution. 
     Then, a voltage of 0 V relative to the reference electrode is applied to the semiconductor electrode. Then, the light source emits a light from the direction of the semiconductor electrode part to the counter electrode part. The light source was a laser light source (Cube 785, Coherent Ltd.) having a wavelength of 785 nm and an intensity of 13 mW. The modulator is thereby excited, electrons released from the excited modulator are transported into the semiconductor layer, and an electric current flows through the semiconductor electrode part and the counter electrode part. This electric current value was measured. 
     An electric current is measured as an electrode-derived electric current without conducting the step of hybridizing the analyte with the probe on the metal layer. The electrode-derived electric current refers to an electric current generated by irradiation of the electrode itself. 
     Comparative Example 3 
     In Comparative Example 3, the same operation as in Example 3 was conducted except that the step of forming the metal layer on the semiconductor layer was not carried out. 
     Comparative Example 4 
     In Comparative Example 4, a silane coupling agent (aminopropyltriethoxysilane: APTES) was used in immobilizing the probe DNA. 
     (Preparation of Semiconductor Electrode Part) 
     Indium tin oxide (ITO) was formed with a thickness of 100 nm as a conductive layer by sputtering on a substrate made of silicon dioxide (SiO 2 ). On this conductive layer, indium oxide (In 2 O 3 ) was formed as a semiconductor layer of 10 nm in thickness by sputtering. This electrode was dipped in a solution having a silane coupling agent (aminopropyltriethoxysilane: PTES) dissolved at a concentration of 1% in toluene, to form a thin film of the silane coupling agent on the semiconductor layer. Then, the electrode was heated at 110° C., then washed with sonication (5 minutes) 3 times in toluene, and washed with dehydrated ethanol, thereby removing the silane coupling agent not bound to the surface of the semiconductor electrode. This conductive layer and the semiconductor layer serve as the semiconductor electrode part. To the semiconductor electrode part was connected the semiconductor electrode lead for connection to an ammeter. 
     (Preparation of Counter Electrode Part) 
     Prepared in the same manner as in Example 3. 
     (Immobilization of Probe Substance) 
     A probe consisting of a DNA (24 bases) is immobilized on the semiconductor layer. First, 6 μL of a solution in which an aqueous solution having a nucleic acid (nucleic acid concentration 100 μM) dispersed therein and an UV crosslinking reagent (Microarray crosslinking reagent D, Amersham) were mixed in a mixing ratio of 1:9 was dropped onto the semiconductor electrode. Thereafter, the resultant was irradiated with UV light (160 mJ) with UV crosslinker (FS-1500, Funakoshi), then washed with ultrapure water and dried for 10 minutes. 
     As a result, the UV crosslinking reagent serves as a crosslinking agent between the DNA and the silane coupling agent, and the nucleic acid is immobilized on the semiconductor layer. 
     (Preparation of Analyte) 
     Prepared in the same manner as in Example 3. 
     (Hybridization Between Analyte and Probe) 
     Performed in the same manner as Example 3. 
     (Preparation of Electrolytic Solution) 
     Prepared in the same manner as Example 3. 
     (Measurement of Photocurrent) 
     Performed in the same manner as in Example 3. 
     (Results) 
       FIG. 15  is a graph of photocurrent values detected in Example 3, Comparative Example 3 and Comparative Example 4. 
     In Example 3, the photocurrent value detected was 158 nA when hybridization was conducted with the DNA having a nucleotide sequence complementary to the probe. When the probe DNA only was immobilized, the photocurrent value detected was 0.082 nA. From this result, S/N=158/0.082=1930. 
     In Comparative Example 3, the photocurrent value detected was 0.24 nA when hybridization was conducted with the DNA having a nucleotide sequence complementary to the probe. When the probe DNA only was immobilized, the photocurrent value detected was 0.028 nA. From this result, S/N=0.24/0.028=8.6. When compared with Example 3, it is revealed that the modulator-derived photocurrent value was 660 times and the S/N ratio was 220 times. 
     In Comparative Example 4, the photocurrent value detected was 19 nA when hybridization was conducted with the DNA having a nucleotide sequence complementary to the probe. When the probe DNA only was immobilized, the photocurrent value detected was 0.021 nA. From this result, S/N=19/0.021=900. When compared with Example 3, it is revealed that the modulator-derived photocurrent value was 8 times and the S/N ratio was 2 times. Similarly to Comparative Example 4, an improvement in modulator-derived photocurrent value and an improvement in S/N ratio are observed. 
     From the foregoing, it is revealed that when a metal layer is formed on the semiconductor electrode part, the detection sensitivity of electric current is improved. The estimated factor for improvement in detection sensitivity of electric current values is that by forming the metal layer, there is brought about (1) increase in the amount of DNA immobilized, (2) improvement in conductivity, and (3) improvement in photoelectric conversion by plasmon excitation in the metal layer. 
     Example 4 
     Verification of Nonspecific Adsorption by Single Nucleotide Polymorphism (SNP) 
     (Preparation of Semiconductor Electrode Part) 
     Indium tin oxide (ITO) was formed with a thickness of 100 nm as a conductive layer by sputtering on a substrate made of silicon dioxide (SiO 2 ). On this conductive layer, indium oxide (In 2 O 3 ) was formed as a semiconductor layer of 10 nm in thickness by sputtering, and a gold thin film was formed as a metal layer of 2 nm in thickness thereon by deposition. The member containing the conductive layer, the semiconductor layer and the metal layer is referred to the semiconductor electrode part. A semiconductor electrode lead for connection to the ammeter is connected to the semiconductor electrode part. 
     (Preparation of Counter Electrode Part) 
     A platinum thin layer formed with a thickness of 200 nm by sputtering on a substrate made of silicon dioxide (SiO 2 ) was used as the counter electrode part. To this counter electrode part was connected a counter lead for connection to the ammeter. 
     (Immobilization of Probe Substance) 
     A probe consisting of a DNA (24 bases) having a thiol group is immobilized on the metal layer of the semiconductor electrode part. First, the semiconductor electrode part is dipped for 18 hours in an aqueous solution having a nucleic acid (nucleic acid concentration 10 μM) dispersed therein. Thereafter, the semiconductor electrode part is washed with ultrapure water and dried for 10 minutes. As a result, the nucleic acid is immobilized on the metal layer by binding a thiol group of the nucleic acid to a gold atom of the metal layer. 
     (Preparation of Analyte) 
     An analyte having a modulator bound to a DNA containing a non-complementary nucleotide sequence (with only 1 non-complementary base) to the probe is prepared. The modulator is Alexa Fluor 750 (Invitrogen). This modulator is an organic dye and is bound to the DNA via a peptide bond. 
     (Hybridization Between Analyte and Probe) 
     The analyte modified with the dye is trapped with the probe on the semiconductor electrode part. 
     First, silicon rubber (thickness 0.2 mm) is arranged as a partition wall around the semiconductor electrode part. 10 μL of a hybridization solution is injected into the space made of this silicon rubber. This hybridization solution was a mixture of the modulator-modified DNA (concentration 10 μM) and a hybridization buffer (Affymetrix). 
     Then, the silicon rubber was covered with a cover glass and subjected to hybridization such that the solution was not dried. Hybridization was carried out by leaving it at 45° C. for 1 hour. After hybridization, the sample was washed with a wash buffer A (Affymetrix) and ultrapure water, and then dried with a blower. 
     (Preparation of Electrolytic Solution) 
     A solvent consisting of acetonitrile (AN) and ethylene carbonate (EC) mixed in a volume ratio of 6:4 is prepared. As a supporting electrolyte salt, tetrapropylammonium iodide (NPr 4 I) is dissolved in an amount of 0.6 M. As an electrolyte, iodine is dissolved in an amount of 0.06 M. The resulting solution is used as the electrolytic solution. 
     (Measurement of Photocurrent) 
     Silicon rubber (thickness 0.2 mm) is arranged as a side wall around the substrate having the semiconductor electrode part where the analyte was hybridized with the probe. 10 μL of the electrolytic solution is injected into the space formed by this silicon rubber. Then, the semiconductor electrode part filled with the electrolytic solution is sealed from above with the substrate having the counter electrode part. The semiconductor electrode part and the counter electrode part are thereby contacted with the electrolytic solution. 
     Then, a voltage of 0 V relative to the reference electrode is applied to the semiconductor electrode. Then, the light source emits a light from the direction of the semiconductor electrode part to the counter electrode part. The light source was a laser light source (Cube 785, Coherent Ltd.) having a wavelength of 785 nm and an intensity of 13 mW. As a result, the modulator is excited, electrons released from the excited modulator are transported into the semiconductor layer, and an electric current flows through the semiconductor electrode part and the counter electrode part. This electric current value was measured. 
     Comparative Example 5 
     In Comparative Example 5, the same operation as in Example 4 was conducted except that a nucleotide sequence complementary to the probe was used. 
     Comparative Example 6 
     In Comparative Example 6, the same operation as in Example 4 was conducted except that the analyte was not hybridized. 
     (Results) 
       FIG. 16  is a graph of photocurrent values detected in Example 4, Comparative Example 5 and Comparative Example 6. 
     In Example 4, the photocurrent value detected was 1.7 nA when hybridization reaction was conducted with the DNA having a non-complementary nucleotide sequence to the probe. In Comparative Example 5, the photocurrent value detected was 195 nA when hybridization reaction was conducted with the DNA having a complementary nucleotide sequence to the probe. When the probe DNA only was immobilized in Comparative Example 6, the photocurrent value detected was 0.067 nA. From this result, it can be confirmed that the amount of DNA adsorbing nonspecifically into the gold thin film is small, and the analyte is detected sequence-specifically. 
     Example 5 
     Dependence of Gold Thin Film on Film Thickness 
     (Preparation of Semiconductor Electrode Part) 
     Indium tin oxide (ITO) was formed with a thickness of 100 nm as a conductive layer by sputtering on a substrate made of silicon dioxide (SiO 2 ). On this conductive layer, indium oxide (In 2 O 3 ) was formed as a semiconductor layer of 10 nm in thickness by sputtering, and a gold thin film was formed as a metal layer of 10 nm in thickness thereon by deposition. The member containing the conductive layer, the semiconductor layer and the metal layer is referred to the semiconductor electrode part. A semiconductor electrode lead for connection to the ammeter is connected to the semiconductor electrode part. 
     (Preparation of Counter Electrode Part) 
     A platinum thin layer was formed with a thickness of 200 nm as a conductive layer by sputtering on a substrate made of silicon dioxide (SiO 2 ), and the resultant was used as the counter electrode part. To this counter electrode part was connected a counter electrode lead for connection to the ammeter. 
     (Immobilization of Probe Substance) 
     A probe consisting of a DNA (24 bases) having a thiol group is immobilized on the metal layer of the semiconductor electrode part. First, the semiconductor electrode part is dipped for 16 hours in an aqueous solution having a nucleic acid (nucleic acid concentration 10 μM) dispersed therein. Thereafter, the semiconductor electrode part is washed with ultrapure water and dried for 10 minutes. As a result, the nucleic acid is immobilized on the metal layer by binding a thiol group of the nucleic acid to a gold atom of the metal layer. 
     (Preparation of Analyte) 
     An analyte having a modulator bound to a DNA containing a complementary nucleotide sequence to the probe is prepared. The modulator is Alexa Fluor 750 (Invitrogen). This modulator is an organic dye and is bound to the DNA via a peptide bond. 
     (Hybridization Between Analyte and Probe) 
     The analyte modified with the dye is trapped with the probe on the semiconductor electrode part. 
     First, silicon rubber (thickness 0.2 mm) is arranged as a partition wall around the semiconductor electrode part. 10 μL of a hybridization solution is injected into the space made of this silicon rubber. This hybridization solution was a mixture of the modulator-modified DNA (concentration 10 μM) and a hybridization buffer (Affymetrix). 
     Then, the silicon rubber was covered with a cover glass and subjected to hybridization such that the solution was not dried. Hybridization was carried out by leaving it at 45° C. for 1 hour. After hybridization, the sample was washed with a wash buffer A (Affymetrix) and ultrapure water, and then dried with a blower. 
     (Preparation of Electrolytic Solution) 
     A solvent consisting of acetonitrile (AN) and ethylene carbonate (EC) mixed in a volume ratio of 6:4 is prepared. As a supporting electrolyte salt, tetrapropylammonium iodide (NPr 4 I) is dissolved in an amount of 0.6 M. As an electrolyte, iodine is dissolved in an amount of 0.06 M. The resulting solution is used as the electrolytic solution. 
     (Measurement of Photocurrent) 
     Silicon rubber (thickness 0.2 mm) is arranged as a side wall around the substrate having the semiconductor electrode part where the analyte was hybridized with the probe. 10 μL of the electrolytic solution is injected into the space formed by this silicon rubber. Then, the semiconductor electrode part filled with the electrolytic solution is sealed from above with the substrate having the counter electrode part. The semiconductor electrode part and the counter electrode part are thereby contacted with the electrolytic solution. 
     Then, a voltage of 0 V relative to the reference electrode is applied to the semiconductor electrode. Then, the light source emits a light from the direction of the semiconductor electrode part to the counter electrode part. The light source was a laser light source (Cube 785, Coherent Ltd.) having a wavelength of 785 nm and an intensity of 13 mW. As a result, the modulator is excited, electrons released from the excited modulator are transported into the semiconductor layer, and an electric current flows through the semiconductor electrode part and the counter electrode part. This electric current value was measured. 
     When the thickness of the gold thin film was changed to 1 nm, 2 nm or 5 nm in preparation of the semiconductor electrode part, electric current values were measured in the same manner as described above. 
     An electric current was measured as an electrode-derived current without conducting the step of hybridizing the analyte with the probe on the metal layer. The electrode-derived current refers to an electric current generated by irradiation of the electrode itself. 
     (Results) 
       FIG. 17  is a graph of S/N ratio in each film thickness detected in Example 5. It can be seen that when the gold thin film is 1 nm, the best S/N ratio can be obtained. 
     When the gold thin film was 5 nm or more, the gold thin film was released from the semiconductor layer by a washing step. Accordingly, when the thickness of the gold thin film was 5 nm or more, the adhesion between the gold thin film and the semiconductor layer should be improved by using a semiconductor layer containing titanium or chrome or by using a semiconductor layer subjected to sintering.