Base sequence detection apparatus and base sequence automatic analyzing apparatus

Abstract

A base sequence detection apparatus is provided with a channel formed on a base sequence detection chip. Working electrodes are formed along the channel and include a probe immobilized thereon, counter electrodes are formed on the inner surface of the channel and, reference electrodes are formed on the inner surface of the channel. An introduction port introduces solution or air from the upstream side of the channel, a delivery port delivers the solution or air in the channel, and the sample is injected into the channel through a sample injection port.

1 - 22 . (canceled)  
   
   
       23 : An analyzing apparatus comprising: 
 a detection unit which detects a target base sequence in a sample solution on the basis of a reaction between the target base sequence in the sample solution and a probe base sequence, including: 
 (a) a substrate on which a channel is formed, the sample solution, a reagent solution, or air being supplied to the channel,  
 (b) a working electrode arranged in the channel with the probe base sequence immobilized thereon,  
 (c) a counter electrode which is arranged in the channel,  
 (d) a reference electrode which is arranged in the channel, and  
 (e) a sealing member which seals the channel on the substrate, which includes inlet and outlet ports communicating with the channel;  
   a supply unit which supplies the reagent solution or air into the channel through the inlet port, which comprises a first nozzle communicated with the inlet port, and a first valve which controls a flow of the reagent solution or air in the first nozzle;    a discharge unit which discharges the sample solution, the reagent solution or air from the channel through the outlet port, and which comprises a second nozzle in communication with the outlet port, a second valve which controls the flow of the reagent solution or air in the second nozzle, and a pump which communicates with the second nozzle and sucks the reagent solution or air from the channel;    a measurement unit which applies a voltage between the working electrode and the counter electrode and detects an electrochemical reaction signal from the working and the counter electrodes;    a temperature control unit which controls a temperature of the detection unit;    a control unit which controls the supply unit, the discharge unit, the measurement unit, and the temperature control unit;    a memory unit which stores the electrochemical reaction signal as measurement data; and    a processing unit which processes the measurement data to determine the target base sequence.    
   
   
       24 : An analyzing apparatus according to  claim 23 , wherein the sealing member includes a sample injection port through which the sample solution is injected into the channel.  
   
   
       25 : An analyzing apparatus according to  claim 23 , wherein the measurement unit comprises: 
 a voltage pattern generator which generates a voltage having a pattern,    a feedback circuit which feeds back a voltage from the reference electrode,    an amplifier which amplifies a voltage which is generated on the basis of the voltages from the voltage pattern generator and the feedback circuit, and applies the amplified voltage to the counter electrode, and    a protective circuit which comprises a resistance arranged in the amplifier or between the amplifier and the counter electrode, and a switch which ON/OFF controls a current that flows to the resistance.    
   
   
       26 : An analyzing apparatus for detecting a target base sequence in a sample on the basis of a reaction between the target base sequence in the sample in a channel and one of probe base sequences, comprising: 
 working electrodes arranged in the channel, which are provided with the probe base sequences immobilized thereon, respectively;    a counter electrode which is arranged in the channel;    a reference electrode which is arranged in the channel;    a voltage pattern generator which generate a voltage having a pattern;    a feedback circuit which feeds back a voltage detected on the reference electrode to the counter electrode;    an amplifier which amplifies the voltage from the voltage pattern generator on the basis of the feedback voltage from the feedback circuit to apply the amplified voltage to the counter electrode;    a resistance connected between the counter electrode and the amplifier; and    a switch which ON/OFF control a current flowing through the resistance.    
   
   
       27 : An analyzing apparatus for detecting a target base sequence in a sample on the basis of a reaction between the target base sequence in the sample in a channel and one of probe base sequences, comprising: 
 working electrodes arranged in the channel, which are provided with the probe base sequences immobilized thereon, respectively;    a counter electrode which is arranged in the channel;    a reference electrode which is arranged in the channel;    a voltage pattern generator which generates a voltage having a pattern;    a feedback circuit which feeds back a voltage detected on the reference electrode to the counter electrode;    an amplifier which amplifies the voltage from the voltage pattern generator on the basis of the feedback voltage from the feedback circuit to apply the amplified voltage to the counter electrode;    a resistance connected to the amplifier in parallel; and    a first switch which ON/OFF control a current flowing through the resistance.    
   
   
       28 : An analyzing apparatus according to  claim 27 , further comprising: 
 a second resistance connected between the counter electrode and the amplifier; and    a high input impedance differential amplifier which amplifies a differential voltage between first and second outputs, the first output being output from a line connecting between the amplifier and the second resistance, and the second output being output from a line connecting between the counter electrode and the second resistance.    
   
   
       29 : An analyzing apparatus according to  claim 27 , further comprising: 
 another reference electrode which is arranged in the channel,    wherein the feedback circuit includes a second high input impedance differential amplifier which differentially amplifies voltages from the reference electrodes and feeds back the amplified differential voltage to the counter electrode.    
   
   
       30 : An analyzing apparatus according to  claim 28 , further comprising: 
 another reference electrode which is arranged in the channel,    wherein the feedback circuit includes a third high input impedance differential amplifier which differentially amplifies voltages from the reference electrodes and feeds back the amplified differential voltage to the counter electrode.    
   
   
       31 : An analyzing apparatus according to  claim 27 , further comprising: 
 a second switch connected between the amplifier and the counter electrode;    a control unit which controls the first and second switches and the voltage pattern generator in a first control state at a first timing, a second control state at a second timing after the first timing, a third control state at a third timing after the second timing, a fourth control state at a fourth timing after the third timing, respectively, wherein    the control unit causes the voltage pattern generator to generate no voltage and maintains the first switch to be closed and the second switch to be opened in the first control state,    the control unit causes the voltage pattern generator to generate no voltage, maintains the first switch to be closed and switches the second switch to be closed in the second control state, and    the control unit causes the voltage pattern generator to generate no voltage, switches the first switch to be opened and maintains the second switch to be closed in the third control state, and    the control unit causes the voltage pattern generator to generate the voltage and maintains the first switch to be opened and the second switch to be closed in the fourth control state so that the voltage is applied to the counter electrode from the voltage generator at the fourth control state.    
   
   
       32 : An analyzing apparatus comprising: 
 a detection unit which detects target base sequences in a sample solution on the basis of a reaction between the target base sequences in a channel and first and second probe base sequences, respectively,    including: 
 (a) a channel formed on the substrate, in which a sample solution, a reagent solution, or air is supplied,  
 (b) first working electrodes arranged in the channel, which are provided with the first probe base sequence immobilized thereon, the first probe base sequence being complementary to a first expected target base sequence having a first base at an SNP position;  
 (c) second working electrodes arranged in the channel, which are provided with the second probe base sequence immobilized thereon, the second probe base sequence being complementary to a second expected target base sequence having a second base at the SNP position;  
 (d) a counter electrode which is arranged in the channel,  
 (e) a reference electrode which is arranged in the channel, and  
 (f) a sealing member which defines the channel on the substrate, which includes inlet and outlet ports communicating with the channel; and  
   a type determination processor which determines a type of the target base sequences at the SNP position, wherein the type determination processor inputs first and second electrochemical signals obtained from the first and second working electrodes, respectively, selects first and second groups of the first and second electrochemical signals, respectively, and tests the first and second groups of first electrochemical signals to determine that the type is a homo type of the first base or the second base, if a significant difference is present between the first and second groups and if the first electrochemical signal is larger, or the second electrochemical signal is larger, respectively, and to determine that the type is a hetero type of the first target base and the second target base, if no significant difference is present.    
   
   
       33 : The analyzing apparatus of  claim 32 , further comprising: 
 an abnormal-data eliminating unit configured to eliminate abnormal data from a data set assigned to the group as a measure of a coefficient of variation.    
   
   
       34 : A base sequence detection unit for detecting a target base sequence in a sample solution on the basis of a reaction between target base sequence in the sample solution in a channel and one of probe base sequences, comprising: 
 a substrate having a major surface;    a sealing member which defines the channel having one and the other ends and extending on the major surface, the sealing member having first and second surfaces, a groove formed on the second surface and first and second ports communicating with the one and the other ends of the channel, respectively, the first and second ports being extended in the sealing member and opened on the sealing member, the second surface being contacted on the major surface and the groove defining the channel;    working electrodes formed on the major surface along the channel, which are provided with the probe base sequences immobilized thereon, respectively;    reference and counter electrodes arranged in the channel;    electrical connecting pads provided on the major surface, which are connected to the working, reference and counter electrodes; and    a cassette body fixing the sealing member on the substrate.    
   
   
       35 : A base sequence detection unit according to  claim 34 , wherein the cassette body comprises a cassette top cover on which the substrate and the sealing member are mounted, and a cassette bottom cover which sandwiches and fixes the substrate and the sealing member together with the cassette top cover.  
   
   
       36 : A base sequence detection unit according to  claim 35 , wherein the cassette top cover comprises an inner surface, an outer surface, a first nozzle insertion hole which extends from the inner surface to the outer surface and is open to the first port, and a second nozzle insertion hole which is open to the second port.  
   
   
       37 : A base sequence detection unit according to  claim 35 , wherein the cassette top cover comprises an inner surface, an outer surface, and an electric connector port which extends from the inner surface to the outer surface and receives the electric connector to electrically connect the electric connector to the pads on the substrate.  
   
   
       38 : A base sequence automatic analyzing apparatus according to  claim 34 , wherein a sectional area of the channel is substantially constant from the first port to the second port.  
   
   
       39 : A base sequence automatic analyzing apparatus according to  claim 34 , wherein a maximum value of the sectional area of the channel is not more than 130% of a minimum value.  
   
   
       40 : A base sequence automatic analyzing apparatus comprising: 
 the base sequence detection unit according to  claim 34 ,    a valve unit including a first valve which switches between a bypass and the channel communicated to the first port, a second valve which switches between the bypass and the channel communicated to the second port, a first nozzle connected to the first valve, and a second nozzle connected to the second valve, wherein the first and second valves and the first and second nozzles are integrally formed;    a probe unit including an electric connector;    a driving unit which drives the valve unit to communicate the first nozzle with the first port, and communicate the second nozzle with the second port, and drive the probe unit so as to connect the pads to the electric connector electrically;    a measurement unit which inputs a measurement signal to the substrate and acquires a numerical signal from the substrate via the probe unit; and    a computer unit which analyzes the numerical signal obtained from the measurement unit.    
   
   
       41 : A base sequence automatic analyzing apparatus according to  claim 40 , wherein one or both of the first and second nozzles have an inner wall having a substantially constant inner diameter and a nozzle lower surface which is substantially perpendicular to a nozzle central axis and substantially flat, and one or both of the first and second ports have a tapered inner wall whose inner diameter gradually increases toward a distal end and a port upper surface which is substantially perpendicular to a port central axis and substantially flat.  
   
   
       42 : A base sequence automatic analyzing apparatus according to  claim 40 , wherein the cassette main body comprises a cassette bottom cover on which the substrate and the sealing member are mounted, and a cassette top cover which sandwiches and fixes the substrate and the sealing member together with the cassette bottom cover.  
   
   
       43 : A base sequence automatic analyzing apparatus according to  claim 42 , wherein the cassette top cover comprises an inner surface, an outer surface, a first nozzle insertion hole which extends from the inner surface to the outer surface and is open to the first port, and a second nozzle insertion hole which is open to the second port.  
   
   
       44 : A base sequence automatic analyzing apparatus according to  claim 42 , wherein the cassette top cover comprises an inner surface, an outer surface, and an electric connector port which extends from the inner surface to the outer surface and receives the electric connector to electrically connects the electric connector to the pads on the substrate.  
   
   
       45 : A base sequence automatic analyzing apparatus according to  claim 42 , wherein a sectional area of the channel is substantially constant from the first port to the second port.  
   
   
       46 : A base sequence automatic analyzing apparatus according to  claim 42 , wherein a maximum value of the sectional area of the channel is not more than 130% of a minimum value.  
   
   
       47 : A base sequence automatic analyzing apparatus according to  claim 42 , further comprising a tube which is connected to the valve unit, and a liquid oscillation mechanism which is arranged on the tube to oscillate the solution in the channel.  
   
   
       48 : A base sequence automatic analyzing apparatus according to  claim 40 , wherein the measurement unit comprises: 
 a voltage pattern generator which generates a voltage pattern to be applied between the working electrode and the counter electrode,    a feedback unit which feeds back a voltage of the reference electrode to the counter electrode,    an amplifier which amplifies the voltage applied to the counter electrode on the basis of a feedback voltage from the feedback unit, and    a protective unit which comprises a resistance arranged in the amplifier or between the amplifier and the counter electrode, and a switch which ON/OFF controls a current that flows to the resistance.    
   
   
       49 : A base sequence automatic analyzing apparatus according to  claim 40 , wherein 
 the apparatus detects a second target base sequence in the sample solution on the basis of a reaction between the second target base sequence in the channel and one of probe base sequences,    the probe base sequences include first and second probe base sequences, the first probe base sequence being complementary to the target base sequence having a first base at an SNP position, and the second probe base sequence being complementary to the target base sequence having a second base at an SNP position, and    the computer comprises a type determination processor which executes type determination processing which includes testing a plurality of first electrochemical signals and a plurality of second electrochemical signals to determine that the target base sequence at the SNP position is a homo type if a significant difference is present between the first and second electrochemical signals, and to determine that the target base sequence at the SNP position is a hetero type if no significant difference is present.