JP5083147B2 - Biopolymer detector - Google Patents

Description

  The present invention relates to a biopolymer detection apparatus using a solid-state imaging device.

  In recent years, we have been analyzing gene expression of various species. Gene expression analysis is to identify a gene expressed in a cell, and specifically, to identify mRNA transcribed from DNA encoding the gene.

  A DNA microarray and its reader have been developed for gene expression analysis. A DNA microarray is obtained by aligning and fixing cDNA having a known base sequence serving as a probe in a matrix on a solid support such as a slide glass (see, for example, Patent Document 1). Here, as a cDNA having a known base sequence, a cDNA having the same base sequence as that of a known mRNA in a specimen or a part thereof is used. Gene expression analysis using a DNA microarray and its reader is performed as follows.

  First, a DNA microarray is prepared in which a plurality of types of cDNAs with known sequences (hereinafter referred to as probe DNA) are aligned and fixed on a solid support such as a slide glass. Next, mRNA is extracted from the specimen, and cDNA labeled with a fluorescent substance (hereinafter referred to as sample DNA) is synthesized using reverse transcriptase. Next, when the sample DNA is labeled with a fluorescent substance and then added to the DNA microarray, the sample DNA is immobilized on the DNA microarray by hybridizing with the complementary probe DNA. When excited, the fluorescent substance that labels the sample DNA emits fluorescence from the position of the probe DNA to which the sample DNA is bound.

Next, the DNA microarray is set in a reader and analyzed by the reader. The reader moves the irradiation point of excitation light two-dimensionally with respect to the DNA microarray, and simultaneously scans the DNA microarray two-dimensionally with a condenser lens and a photomultiplier. The fluorescence emitted from the fluorescent material excited by the excitation light is collected by a condensing lens, and the fluorescence intensity is measured by a photomultiplier to measure the fluorescence intensity distribution in the surface of the DNA microarray. The fluorescence intensity distribution on the microarray is output as a two-dimensional image. In the output image, the portion having high fluorescence intensity indicates that sample DNA having a base sequence complementary to the base sequence of the probe DNA is included. Therefore, it is possible to identify which mRNA of the known sequence is expressed in the specimen depending on which part of the two-dimensional image has high fluorescence intensity. Moreover, it is possible to determine whether a tumor cell is sensitive to an anticancer drug by diagnosing whether or not the patient is afflicted with a disease using the quantitative value of mRNA as an index (Patent Literature). 2, 3).
Japanese Patent Laid-Open No. 2002-286643 Japanese Patent No. 3507884 Japanese Patent No. 4058263

  Incidentally, the inventors have developed a biopolymer detection chip in which probe DNA is spotted on the light receiving surface of a solid-state imaging device. In such a biopolymer detection chip, the address of the portion where the amount of received light is relatively large in the image data acquired by the solid-state imaging device corresponds to the position of the spot to which the sample DNA is attached. Therefore, by specifying the address of the portion where the amount of received light is large, it is possible to specify to which spot of the plurality of spots the sample DNA is attached, and to specify the sequence of the sample DNA. In such a biopolymer detection chip, since spots are scattered on the light receiving surface of the solid-state imaging device, there is an advantage that a lens or the like is not required and the apparatus is downsized.

The solid-state imaging device is a two-dimensional array in which a plurality of photoelectric conversion elements that linearly convert an incident light amount into an electric signal and output the electric signal. Within the dynamic range of the solid-state imaging device, it is preferable that the incident light amount and the output value of the photoelectric conversion element have a linear function relationship. However, the range in which the incident light amount and the output value have a linear function relationship is narrow, and the dynamic range of the solid-state imaging device is narrow. In addition, in order to widen the dynamic range, a table showing the relationship between the incident light quantity and the output value of the photoelectric conversion element in detail was created to determine the incident light quantity, but a storage device for storing a large amount of data is necessary. There was a problem of being.
Therefore, the present invention is to enable a good determination without requiring a large storage device even when the incident light amount and the output value are not in a linear function relationship.

In order to solve the above problems, according to the present invention, in the biopolymer detection apparatus,
A solid-state imaging device having a light receiving surface and a plurality of light receiving elements arranged below the light receiving surface;
A plurality of types of spots made of known biopolymers and scattered on the light receiving surface of the solid-state imaging device,
A driving circuit capable of outputting an output value corresponding to the amount of incident light of the light receiving element during the optical sensing time in which photoelectric conversion is performed by the light receiving element by driving the solid-state imaging device, and changing the light sensing time; ,
A controller that obtains a weighted average of the output value of the light receiving element when the optical sensing time is the first time and the output value of the light receiving element when the optical sensing time is the second time. And
It is preferable that the controller obtains the weighted average such that a deviation rate between the weighted average and the linear approximation obtained from the weighted average is −5% to 5%.
It is preferable that the controller obtains a linear approximation regression equation from the weighted average.
It is preferable that the controller obtains the incident light quantity of the light receiving element based on a linear approximation regression equation obtained from the weighted average.
It is preferable to have a storage device that stores the slope and intercept of the regression equation of linear approximation from the weighted average.
An inclination and an intercept are read from the storage device, and an incident light amount value is obtained from the weighted average value at the time of measurement.
By measuring the incident light amount value of the fluorescent substance corresponding to the standard molecular weight in advance and setting it as a standard value, the mRNA amount can be quantified by the ratio between the incident light amount value and the standard incident light amount value. And

  According to the present invention, the weighted average based on the output value of the photoelectric conversion element when the optical sensing time is set to the first time and the output value of the photoelectric conversion element when the optical sensing time is set to the second time From this, a regression equation of linear approximation can be obtained, and a good judgment can be made.

  Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. However, although various technically preferable limitations for implementing the present invention are given to the embodiments described below, the scope of the invention is not limited to the following embodiments and illustrated examples.

[1] Outline of Biopolymer Detection Device FIG. 1 is a perspective view showing a main part of a biopolymer detection device 80 in an embodiment of the present invention.
As shown in FIG. 1, in this biopolymer detection device 80, the biopolymer detection chip 1, the cooling device 2, and the temperature sensor 3 are mounted on a heat transfer table 4. An excitation light irradiation device 81 is disposed above the heat transfer table 4.

  The biopolymer detection chip 1 includes a solid-state imaging device 10, spots 60, 60,..., A drive circuit 70, and a storage device 82.

The solid-state imaging device 10 is installed on the heat transfer table 4 with good thermal conductivity with its light receiving surface facing the excitation light irradiation device 81. The solid-state imaging device 10 is removable from the heat transfer table 4.
The spots 60, 60,... Are spotted on the light receiving surface of the effective pixel region 11 of the solid-state imaging device 10. The spots 60, 60,... Are arranged in a matrix.
The drive circuit 70 and the storage device 82 are mounted in the area 12 around the effective pixel area 11. A flexible wiring sheet 83 is connected to the drive circuit 70 and the storage device 82.

[2] Solid-State Imaging Device The solid-state imaging device 10 will be described with reference to FIGS. FIG. 2 is an enlarged plan view of one spot 60 in FIG. FIG. 3 is a plan view showing the light receiving element 20 including one double gate transistor which is a photoelectric conversion element for receiving the fluorescence of the labeled fluorescent substance remaining in the spot 60 by the hybridization. 4 is a cross-sectional view taken along the line IV-IV shown in FIG.

  As shown in FIG. 2, the solid-state imaging device 10 has a plurality of light receiving elements 20, 20,. The solid-state imaging device 10 is provided with a plurality of bottom gate lines 41, source lines 42, drain lines 43, and top gate lines 44.

  As shown in FIG. 4, the solid-state imaging device 10 includes a transparent substrate 13, a bottom gate insulating film 22, a top gate insulating film 29, a protective insulating film 32, an excitation light shielding film 33, and a spot fixing layer 34. Are stacked.

  The transparent substrate 13 has a property of transmitting light (hereinafter referred to as “light transmissive”) and an insulating property, and is a glass substrate (for example, a quartz glass substrate), a plastic substrate (for example, made of polycarbonate or PMMA). Substrate) Other insulating transparent substrate.

The bottom gate insulating film 22, the top gate insulating film 29, and the protective insulating film 32 have insulating properties and light transmissive properties, and are, for example, a silicon nitride film or a silicon oxide film.
The excitation light shielding film 33 shields excitation light (for example, light in the ultraviolet wavelength region) emitted from the excitation light irradiation device 81 and transmits fluorescence (for example, light in the visible light wavelength region) having a wavelength different from that of the excitation light. It has the property to do.
The spot fixing layer 34 fixes the spot 60 by covalently bonding the probe to be the spot 60 with a silane coupling agent or the like.

  In the solid-state imaging device 10, the light receiving element 20 is used as a photoelectric conversion element, and the light receiving element 20 is a pixel. A plurality of light receiving elements 20, 20,... Are arranged on the transparent substrate 13 in a two-dimensional array, particularly in a matrix. The address of the light receiving element 20 in the first row and the first column is set to (1, 1), and the address of the light receiving element 20 in the nth row and the mth column is set to (n, m). These light receiving elements 20, 20,... Are collectively covered with a protective insulating film 32. 3 shows 64 light receiving elements 20, 20,... In 8 rows × 8 columns, but the number of light receiving elements 20 is not limited to 64 as long as it is plural.

  As shown in FIGS. 3 and 4, the light receiving element 20 is a double gate transistor, and includes a bottom gate electrode 21, a bottom gate insulating film 22, a semiconductor film 23, a channel protective film 24, an impurity semiconductor film 25, and an impurity semiconductor film. 26, a source electrode 27, a drain electrode 28, a top gate insulating film 29, and a top gate electrode 31.

  The bottom gate electrode 21 is formed between the transparent substrate 13 and the bottom gate insulating film 22. The semiconductor film 23, the channel protective film 24, the impurity semiconductor film 25, the impurity semiconductor film 26, the source electrode 27 and the drain electrode 28 are formed between the bottom gate insulating film 22 and the top gate insulating film 29. The top gate electrode 31 is formed between the top gate insulating film 29 and the protective insulating film 32.

  The bottom gate electrode 21 is formed on the transparent substrate 13 for each light receiving element 20. In addition, a plurality of bottom gate lines 41, 41,... Are formed between the transparent substrate 13 and the bottom gate insulating film 22, and the bottom gate lines 41, 41,. (Horizontal direction). The bottom gate electrodes 21 of the light receiving elements 20, 20,... In the same row arranged in the row direction are formed integrally with a common bottom gate line 41. The bottom gate electrode 21 and the bottom gate line 41 have conductivity and light shielding properties, and are made of, for example, chromium, a chromium alloy, aluminum, an aluminum alloy, or an alloy thereof.

  The semiconductor film 23 is opposed to the bottom gate electrode 21 with the bottom gate insulating film 22 interposed between the semiconductor film 23 and the bottom gate electrode 21. The semiconductor film 23 is formed independently for each light receiving element 20. The semiconductor film 23 is a light receiving portion, and an amount of electron-hole pairs corresponding to the amount of light incident on the semiconductor film 23 is formed in the semiconductor film 23. The semiconductor film 23 is made of amorphous silicon.

  The channel protective film 24 is formed on the central portion of the semiconductor film 23. The channel protective film 24 is independently patterned for each light receiving element 20. The channel protective film 24 has insulating properties and light transmissive properties, and is made of, for example, silicon nitride or silicon oxide. The channel protective film 24 protects the semiconductor film 23 from an etchant used for patterning. When light enters the semiconductor film 23, holes in the electron-hole pair carriers generated in the semiconductor film 23 are accumulated near the interface between the channel protective film 24 and the semiconductor film 23.

The impurity semiconductor film 25 is formed so as to overlap a part of the semiconductor film 23. Part of the impurity semiconductor film 25 overlaps the channel protective film 24. The impurity semiconductor film 26 is formed so as to overlap another part of the semiconductor film 23. Part of the impurity semiconductor film 26 overlaps with the channel protective film 24. The impurity semiconductor films 25 and 26 are patterned independently for each light receiving element 20. The impurity semiconductor films 25 and 26 are made of amorphous silicon (n ⁺ silicon) containing n-type impurities (for example, phosphine).

  The source electrode 27 overlaps the impurity semiconductor film 25. The drain electrode 28 overlaps the impurity semiconductor film 26. The source electrode 27 and the drain electrode 28 are formed for each light receiving element 20. A plurality of source lines 42, 42,... And drain lines 43, 43,... Are formed between the bottom gate insulating film 22 and the top gate insulating film 29. The source lines 42, 42,. 43 extend in the column direction (vertical direction). The source electrodes 27 of the light receiving elements 20, 20,... Arranged in the column direction are formed integrally with the common source line 42, and the light receiving elements 20 in the same column arranged in the column direction. , 20,... Are formed integrally with a common drain line 43. The source electrode 27, the drain electrode 28, the source line 42, and the drain line 43 have conductivity and light shielding properties, and are made of, for example, chromium, a chromium alloy, aluminum, an aluminum alloy, or an alloy thereof.

  The top gate electrode 31 is opposed to the semiconductor film 23 with the channel protective film 24 and the top gate insulating film 29 sandwiched between the top gate electrode 31 and the semiconductor film 23. The top gate electrode 31 is formed on the top gate insulating film 29 for each light receiving element 20. Further, a plurality of top gate lines 44, 44,... Are formed between the top gate insulating film 29 and the protective insulating film 32, and these top gate lines 44, 44,. It extends to. The top gate electrodes 31 of the light receiving elements 20, 20,... In the same row arranged in the row direction are formed integrally with the common top gate line 44. The top gate electrode 31 and the top gate line 44 are conductive and light transmissive, for example, indium oxide, zinc oxide, tin oxide, or a mixture containing at least one of them (for example, tin-doped indium oxide ( ITO) and zinc-doped indium oxide).

  The solid-state imaging device 10 configured as described above uses the surface 35 of the spot fixing layer 34 as a light-receiving surface, and the amount of light incident on the semiconductor film 23 of the light-receiving element 20 when the solid-state imaging device 10 is driven becomes an electrical signal. Converted.

5 and 6 are graphs showing the photosensitive characteristics of the light receiving element 20.
In FIG. 5, the horizontal axis represents the optical sensing time (unit: second), and the vertical axis represents the output value converted based on the output voltage to the drain line 43 by photoelectric conversion of the light receiving element 20 (unit: arbitrary unit). The output value increases as the amount of light received by the light receiving element 20 increases. In FIG. 5, the amount of light incident on the light receiving element 20 is 1.0 mcd, 0.5 mcd, 0.2 mcd, or 0.0 mcd. The optical sensing time is a so-called exposure time. That is, the optical sensing time is a time during which light is irradiated to the light receiving element 20, carriers are accumulated in the light receiving element 20, and photoelectric conversion is performed in the light receiving element 20.
In FIG. 6, the horizontal axis represents the amount of light incident on the light receiving element 20 (unit: mcd), and the vertical axis represents the output value (unit) converted from the output voltage to the drain line 43 of the light receiving element 20 by the amount of incident light. : Arbitrary unit). In FIG. 6, the optical sensing time was 1 second and 3 seconds, respectively. In the present embodiment, the first time is 1 second and the second time is 3 seconds.

As is clear from FIG. 6, when the optical sensing time is 1 second or 3 seconds, the line representing the relationship between the incident light amount and the output value is a curve, so the output value is not a linear function of the incident light amount. The linear approximation regression equation (1) obtained from the weighted average which is a value obtained by dividing the optical sensing time by 3 times the output value at 1 second and the output value at 3 seconds by 4,
Output value = 19.591 × incident light quantity (mcd) −9.4673. . . (1)
It becomes. The deviation rate of the weighted average with respect to the regression equation of linear approximation at the slope of 19.591 and the intercept of −9.4673 was in the range of 2.4% to −4.4%. The deviation rate is preferably within the range of −5% to 5%. Therefore, the amount of incident light can be measured by substituting the output value from the linear approximation regression equation. Specifically, the controller 84 described later measures an output value read at a light sensing time of 1 second and an output value read at a light sensing time of 3 seconds at the light receiving element 20 located at a certain spot 60. 3 times the output value of the optical sensing time of 1 second, add the output value of the optical sensing time of 3 seconds, and calculate the weighted average of the output value divided by 4, and the weighted average of this output value is The amount of incident light can be obtained by substituting it into the output value of equation (2). In addition, the molecular weight of mRNA that has been determined in advance is used as the standard molecular weight, and the amount of fluorescence emitted by the molecular weight of the corresponding fluorescent substance is measured with this solid-state imaging device. It can be obtained based on the ratio between the light quantity and the standard incident light quantity.
Incident light quantity = (output value + 9.4673) /19.591. . . (2)
The mRNA amount can also be determined by substituting the target incident light amount for the incident light amount in the following formula (3).
The amount of mRNA = (the amount of incident light / the amount of standard incident light) × standard molecular weight. . . (3)
According to the above equation (3), the mRNA amount is determined with an accuracy of 2.4% to -4.4%.
Here, when the signal accumulation time is 1 second, the weight coefficient of the output value is 3, and when the signal accumulation time is 3 seconds, the weight coefficient of the output value is 1, and the weighted average is obtained as follows.
Weighted average = {(output value when signal accumulation time is 1 second) × 3 + (output value when signal accumulation time is 3 seconds) × 1} / (3 + 1)

  By regression analysis (least square method), a regression equation of linear approximation is obtained from this weighted average curve, and a slope (proportional coefficient) and an intercept are obtained. The inclination and the intercept obtained in advance in this way are recorded in the storage device 82.

  FIG. 7 shows an output value when the optical sensing time is 1 second, an output value when the optical sensing time is 3 seconds, a weighted average thereof, a linear approximation when the incident light quantity is 0.2 mcd, 0.5 mcd, and 1.0 mcd. It shows the deviation rate between the weighted average and linear approximation.

[3] Spots As shown in FIGS. 1 and 2, spots 60, 60,... Are formed on the light receiving surface 35 of the solid-state imaging device 10. The spot 60 may be a crowd of cDNAs (probe DNAs) having a known base sequence or a crowd of known antibodies. The spot 60 is formed by dropping a solution of cDNA (probe DNA) serving as a probe, an antibody or other biopolymer onto the light receiving surface 35 of the solid-state imaging device 10 and drying it. Hereinafter, a case where cDNA having a known base sequence is used as a probe will be described.

  In one spot 60, a large number of single-stranded probe DNAs having the same base sequence are gathered and the cluster is immobilized. The base sequence of the probe DNA is different for each spot 60. As the probe DNA, a known mRNA base sequence is used, or a DNA having the same or complementary base sequence as a part thereof is used. Specifically, for example, a cDNA library prepared from the same cell specimen as that used for fluorescently labeled DNA can be used.

  As shown in FIG. 2, one spot 60 is formed so as to overlap a plurality of light receiving elements 20. Note that the number of spots 60 spotted on the light receiving surface 35 of the solid-state imaging device 10 may be any number. In addition, the number of light receiving elements 20 that overlap one spot 60 may be any number.

[4] Light Shielding As shown in FIG. 1, a light shielding object 65 is provided on a part of the light receiving surface 35 of the solid-state imaging device 10. The light shield 65 shields light. Accordingly, excitation light, fluorescence, or the like does not enter the light receiving element 20 below the light shield 65.

[5] Drive Circuit FIG. 8 is a circuit diagram showing the solid-state imaging device 10 and its peripheral circuits.
The drive circuit 70 shown in FIG. 1 includes a top gate driver 71, a bottom gate driver 72, and a drain driver 73 as shown in FIG.

  Are connected to the terminals of the top gate driver 71, the bottom gate lines 41, 41,... Are connected to the terminals of the bottom gate driver 72, and the drain lines 43, 43,. Is connected to the terminal of the drain driver 73. Further, the source lines 42, 42,... Of the solid-state imaging device 10 are connected to a constant voltage source, and in this example, the source lines 42, 42,. The top gate driver 71 is a first scanning driver that scans the top gate lines 44, 44,... In the column direction. The bottom gate driver 72 is a second scanning driver that scans the bottom gate lines 41, 41,... In the column direction.

FIG. 9 is a timing chart showing a transition of a signal for driving the solid-state imaging device 10.
The top gate driver 71 selects and scans the light receiving elements 20, 20,... Line-sequentially by sequentially selecting the top gate lines 44, 44,. Specifically, as shown in FIG. 9, the top gate driver 71 sequentially outputs reset pulses for eliminating holes accumulated in the respective light receiving elements 20 to the top gate lines 44, 44,. The level of the reset pulse is a high level of +5 [V]. On the other hand, the top gate driver 71 applies a low level potential of −20 [V] to each top gate line 44 when no reset pulse is output. As the top gate driver 71, a shift register can be used. Due to this negative potential, holes in the electron-hole pair are electrically trapped in the electron-hole pair generated in the semiconductor film 23.

  The bottom gate driver 72 selects and scans the light receiving elements 20, 20,... In a line-sequential manner by sequentially selecting the bottom gate lines 41, 41,. Specifically, as shown in FIG. 9, the bottom gate driver 72 sequentially outputs read pulses to the bottom gate lines 41, 41,. The level of the read pulse is +10 [V] on level (high level), and the level when the read pulse is not output is ± 0 [V] off level (low level). As the bottom gate driver 72, a shift register can be used.

  The horizontal scanning period of the top gate driver 71 (the period from when a reset pulse is output to an arbitrary row until the reset pulse is output to the next row), and the horizontal scanning cycle of the bottom gate driver 72 (read pulse at an arbitrary row) And the period from when the read pulse is output to the next row). The rising edge of the read pulse by the bottom gate driver 72 is shifted after the falling edge of the reset pulse by the top gate driver 71. During this period, the light receiving element 20 accumulates holes in an amount corresponding to the amount of incident light. Optical sensing time.

  Specifically, after the top gate driver 71 outputs a reset pulse to the top gate line 44 of any row, the bottom gate driver 72 outputs a read pulse to the bottom gate line 41 of the same row after a light sensing time. Further, the top gate driver 71 and the bottom gate driver 72 shift the output signal.

  The drain driver 73 reads the output values of the light receiving elements 20, 20,... Read by the top gate driver 71, and sequentially outputs the read output values of the light receiving elements 20, 20,. Specifically, as shown in FIG. 9, the drain driver 73 precharges all drain lines 43, 43,... Before the read pulse is output after the reset pulse of each row is output. A charge pulse is output. The level of the precharge pulse is a high level of +5 [V], and the level when the precharge pulse is not output is a low level of ± 0 [V]. The drain driver 73 outputs the voltages (output values) of the drain lines 43, 43,... In order of columns after outputting the precharge pulse. Thereby, an image is input by the solid-state imaging device 10.

  Further, the top gate driver 71 sequentially selects the top gate line 44 from the first row to the last row as described above a plurality of times, and the bottom gate driver 72 performs the final selection from the first row as described above. The bottom gate line 41 in the row is sequentially selected a plurality of times. Therefore, image input is performed a plurality of times by the solid-state imaging device 10.

  Here, since the solid-state imaging device 10 obtains a weighted average that is more linear with respect to the amount of incident light, a plurality of image inputs with different optical sensing times are performed. The controller 84 sends a clock signal or the like to the top gate driver 71 and the bottom gate driver 72 so as to change the optical sensing time between the reset pulse falling timing by the top gate driver 71 and the rising edge of the read pulse by the bottom gate driver 72. A control signal is output. Specifically, image input is performed twice, with an optical sensing time of 1 second and 3 seconds.

[6] Storage Device The storage device 82 shown in FIG. 1 is a hard disk drive or a semiconductor storage device.
In the storage device 82, the slope and intercept of Equation (1) obtained in advance are recorded. Hereinafter, the inclination recorded in the storage device 82 is A, and the intercept recorded in the storage device 82 is B.
As described above, the slope A is a slope of a linear function obtained from the regression equation for the relationship between the weighted average of output values and the amount of incident light at different optical sensing times, and the intercept B is an intercept of the linear function. When the linear function is expressed by an equation, “y = Ax + B” is obtained. In the graph shown in FIG. 6, A is 195.91 and B is −9.4673.

[7] Heat Transfer Table The heat transfer table 4 shown in FIG. 1 is made of a material having a high thermal conductivity, specifically, iron, stainless steel, or other metal material.

[8] Cooling device The cooling device 2 shown in FIG. 1 cools the heat transfer table 4 by forcibly releasing the heat of the heat transfer table 4 to the outside, specifically, a Peltier element, Heat pump and other cooling devices.

[9] Temperature Sensor The temperature sensor 3 shown in FIG. 1 detects the temperature of the solid-state imaging device 10 and converts the detected temperature into an electrical signal. Specifically, the thermistor, thermocouple, or other temperature sensor. It is. Here, the temperature sensor 3 is mounted on the heat transfer table 4, and the temperature of the heat transfer table 4 is detected by the temperature sensor 3. Since the heat conductivity of the heat transfer table 4 is high, the solid-state imaging device 10 The temperature is detected by the temperature sensor 3 with almost no delay. The temperature sensor 3 may be provided in the solid-state imaging device 10.

[10] Other Components FIG. 10 is a block diagram showing the configuration of the biopolymer detection device 80.
In addition to the biopolymer detection chip 1, the cooling device 2, the temperature sensor 3, the heat transfer table 4, and the excitation light irradiation device 81, the biopolymer detection device 80 includes a controller 84, an output device 85, a memory 86, and A / D conversion. A container 87 is provided.

  The storage device 82, the top gate driver 71, the bottom gate driver 72, and the drain driver 73 are connected to the controller 84 by the flexible wiring sheet 83 shown in FIG. The output terminal of the drain driver 73 is connected to the A / D converter 87 by the flexible wiring sheet 83.

The controller 84 has a function of turning on / off the excitation light irradiation device 81.
The controller 84 has a function of setting the optical sensing time by setting the horizontal scanning period of the top gate driver 71 and the bottom gate driver 72.
The controller 84 outputs control signals to the top gate driver 71, the bottom gate driver 72, and the drain driver 73 in a state where the excitation light irradiation device 81 is turned on, so that the top gate driver 71 and the bottom gate driver are set in the set light sensing time. 72 and a function of operating the drain driver 73. Thereby, the imaging operation of the solid-state imaging device 10 is performed.

  The A / D converter 87 quantizes the output of the drain driver 73 (the output value of each light receiving element 20) and outputs it to the controller 84.

  The controller 84 has a function of recording the output of the A / D converter 87 in the memory 86. Thereby, the output value distribution along the light receiving surface 35 of the solid-state imaging device 10 is recorded in the memory 86 as two-dimensional image data.

  Here, the controller 84 operates the top gate driver 71, the bottom gate driver 72, and the drain driver 73 to perform image input twice with different optical sensing times. For the first time, the controller 84 sets the optical sensing time to 1 second, and operates the top gate driver 71, the bottom gate driver 72, and the drain driver 73 with the optical sensing time. For the second time, the controller 84 sets the optical sensing time to 3 seconds and operates the top gate driver 71, the bottom gate driver 72, and the drain driver 73 during the optical sensing time. As a result, the first and second image data are recorded in the memory 86, respectively.

  The controller 84 triples the output value of the light receiving element 20 at each address for the first time, adds the output value of the light receiving element 20 at the second address for the second time, and divides the output value by 4. Calculate the weighted average. Using the slope A and the intercept B recorded in the storage device 82, the weighted average of the output values of the light receiving elements 20 at the respective addresses is substituted into the output values of the expression (2), and the incident light quantity of the light receiving elements 20 at the respective addresses is determined. Each is obtained and stored in the memory 86.

  The controller 84 has a function of causing the output device 85 to output an image based on each address recorded in the memory 86 and the amount of incident light at each address. The output device 85 is, for example, a plotter, a printer, or a display.

Further, since the output value of each light receiving element 20 is temperature-dependent, the controller 84 sets the set temperature at which each light receiving element 20 maintains the characteristics in the ranges of FIGS. 5 and 6 with respect to the temperature control circuit 88. Has the function of setting.
The temperature control circuit 88 controls the cooling device 2. Here, the temperature control circuit 88 performs constant temperature control to keep the set temperature by feeding back the temperature detected by the temperature sensor 3 and controlling the cooling device 2 based on the detected temperature. For example, when lower threshold value <set temperature <upper threshold value, when the detected temperature is lower than the lower threshold value, the temperature control circuit 88 increases the cooling capacity of the cooling device 2 and the detected temperature is equal to or higher than the lower threshold value and lower than the upper threshold value. Sometimes, the temperature control circuit 88 maintains the cooling capacity of the cooling device 2, and when the detected temperature exceeds the upper threshold, the temperature control circuit 88 decreases the cooling capacity of the cooling device 2.

[11] Analysis Procedure A method for analyzing a sample using the biopolymer detector 80 will be described, and a method for using and operating the biopolymer detector 80 will be described.

[11-1] Preparation of fluorescence-labeled DNA DNA can be used as a sample to be analyzed. As a sample DNA, mRNA obtained by extracting mRNA expressed in an arbitrary cell specimen and performing RT-PCR reaction using reverse transcriptase can be used. The cDNA is labeled with a fluorophore. As the phosphor, for example, Cy2 (absorption wavelength 491 nm, fluorescence wavelength 509 nm), Cy3 (absorption wavelength 553 nm, fluorescence wavelength 569 nm), Cy5 (absorption wavelength 645 nm, fluorescence wavelength 664 nm) manufactured by GE Healthcare Bioscience Co., Ltd. are used. be able to.

  In order to label cDNA with a phosphor, for example, an RT-PCR reaction may be performed using an oligo dT primer labeled with a phosphor or a labeled dNTP mix. Hereinafter, this labeled cDNA is referred to as fluorescently labeled DNA.

[11-2] Hybridization First, a solution containing fluorescently labeled DNA (hereinafter, fluorescently labeled DNA) is applied to the light receiving surface 35 of the solid-state imaging device 10. The fluorescently labeled DNA solution may be dropped on each of the spots 60, 60,.

  At this time, the fluorescently labeled DNA solution is heated so that the DNA becomes a single strand. Further, the light receiving surface 35 of the solid-state imaging device 10 is also heated so that the DNA of the spot 60 becomes a single strand.

  Next, the solid-state imaging device 10 is cooled. Then, of the fluorescently labeled DNA in the fluorescently labeled DNA solution, the fluorescently labeled DNA complementary to the probe DNA of the spot 60 hybridizes with the probe DNA. On the other hand, fluorescently labeled DNA that is not complementary to the probe DNA does not bind to the spot 60, or even if it binds to the spot 60.

  Thereafter, the fluorescently labeled DNA solution is washed away from the light receiving surface 35 of the solid-state imaging device 10 with a washing buffer solution, and the fluorescently labeled DNA that has not been hybridized is removed from the light receiving surface 35 of the solid-state imaging device 10.

[11-3] Detection of Sample After performing the above processing, the biopolymer detection chip 1 is placed on the heat transfer table 4 and the flexible wiring sheet 83 is connected to the controller 84. Thereby, the top gate driver 71, the bottom gate driver 72, the drain driver 73, and the storage device 82 are connected to the controller 84, and the drain driver 73 is connected to the A / D converter 87. Thereafter, the controller 84 is activated.

  First, the controller 84 sets a set temperature (for example, 10 ° C.) for the temperature control circuit 88 after startup. Then, the temperature control circuit 88 controls the cooling device 2 while feeding back the temperature detected by the temperature sensor 3. Thereby, the heat-transfer base 4 and the solid-state imaging device 10 become preset temperature, and are maintained at the preset temperature. Since the solid-state imaging device 10 is cooled by the cooling device 2 and the heat transfer table 4, the dark current of the solid-state imaging device 10 is reduced.

  Next, the controller 84 turns on the excitation light irradiation device 81. The spots 60 and 60 are irradiated by the excitation light irradiation device 81. Then, fluorescence is emitted from the spot 60 where the fluorescently labeled DNA is bound to the probe DNA. On the other hand, no fluorescence is emitted from the spot 60 where the fluorescently labeled DNA is bound to the probe DNA.

  Then, the controller 84 outputs a control signal to the top gate driver 71, the bottom gate driver 72, and the drain driver 73 so that the light sensing time becomes 1 second with the excitation light irradiation device 81 turned on. Then, the top gate driver 71, the bottom gate driver 72, and the drain driver 73 operate with an optical sensing time of 1 second, and image input by the solid-state imaging device 10 is performed.

  An image input operation of the solid-state imaging device 10 by the top gate driver 71, the bottom gate driver 72, and the drain driver 73 will be specifically described with reference to FIG.

  The top gate driver 71 sequentially outputs reset pulses to the top gate line 44 from the first row to the last row at a predetermined horizontal scanning period, and the bottom gate driver 72 rises one second after the falling timing of the reset pulse. Read pulses are sequentially output from the first row to the bottom gate line 41 of the last row at a predetermined horizontal scanning period. At that time, the drain driver 73 outputs a precharge pulse to all the drain lines 43, 43,... Between the reset period in which the reset pulse is output in each row and the period in which the read pulse is output in each row. .

  The operation of each light receiving element 20 in the i-th row will be described in detail. When the top gate driver 71 outputs a reset pulse to the i-th top gate line 44, the i-th top gate line 44 goes to a high level. While the i-th top gate line 44 is at a high level (this period is referred to as a reset period), in each light-receiving element 20 in the i-th row, the semiconductor film 23, the semiconductor film 23, the channel protective film 24, Carriers accumulated in the vicinity of the interface (here, holes) are repelled and discharged by the voltage of the top gate electrode 31.

  Then, the top gate driver 71 ends outputting the reset pulse to the i-th top gate line 44, and the fluorescence is incident on the semiconductor film 23 to generate electron-hole pairs generated in the semiconductor film 23. A negative potential (−20 [V]) is output to the top gate line 44 in order to electrically capture the positive holes. That is, in the optical sensing time from the end of the reset pulse of the i-th top gate line 44 to the output of the read pulse to the i-th bottom gate line 41, an amount of electron-positive in accordance with the amount of light. The hole pairs are generated in the semiconductor film 23, and the holes are accumulated in the semiconductor film 23 or in the vicinity of the interface between the semiconductor film 23 and the channel protective film 24 by the electric field of the top gate electrode 31.

  During the optical sensing time, the drain driver 73 outputs a precharge pulse to all the drain lines 43, 43,. While the precharge pulse is output (referred to as a precharge period), in each light receiving element 20 in the i-th row, the potential applied to the top gate electrode 31 is −20 [V]. Then, the electric field due to the holes accumulated in the semiconductor film 23 or in the vicinity of the interface between the semiconductor film 23 and the channel protective film 24 due to the negative electric field inevitably cancels the negative electric field completely, so that the channel region of the semiconductor film 23 is obtained. Therefore, it cannot be a positive electric field that can form an n-channel. Then, since the potential applied to the bottom gate electrode 21 is ± 0 [V], a channel is formed in the semiconductor film 23 even if a potential difference of the precharge pulse occurs between the drain electrode 28 and the source electrode 27. In other words, no current flows between the drain electrode 28 and the source electrode 27. Since no current flows between the drain electrode 28 and the source electrode 27 in the precharge period, the precharge pulse output to the drain lines 43, 43,... Charge is charged.

  The drain driver 73 finishes outputting the precharge pulse, and the bottom gate driver 72 outputs a read pulse to the i-th bottom gate line 41. While the bottom gate driver 72 outputs a read pulse to the i-th bottom gate line 41 (this period is referred to as a read period), the bottom gate electrode 21 of each light-receiving element 20 in the i-th row is +10 [ Since the potential V] is applied, each light receiving element 20 in the i-th row is turned on.

  In the lead period, the carriers accumulated during the optical sensing time work to alleviate the negative electric field of the top gate electrode 31. Therefore, if the amount of incident light is sufficient and the amount of carriers is sufficient, an n-channel is formed in the semiconductor film 23 together with the positive electric field of the bottom gate electrode 21, and current flows from the drain electrode 28 to the source electrode 27. Flows, and the charge of the drain electrode 28 decreases. Therefore, in the read period, the voltages of the drain lines 43, 43,... Tend to gradually decrease with time due to the drain-source current.

  As the amount of light incident on the semiconductor film 23 in the light sensing time increases, the number of accumulated carriers also increases, and as the number of accumulated carriers increases, the charge reduction rate of the drain electrode 28 in the read period increases and the read period The level of the current flowing from the drain electrode 28 to the source electrode 27 in FIG. Therefore, the decreasing tendency of the voltage of the drain lines 43, 43,... During the lead period is deeply related to the amount of light incident on the semiconductor film 23 during the optical sensing time.

  .., And the voltage (output value) of the drain lines 43, 43,... After a lapse of a predetermined time from the start of the read period between the i-th read period and the next (i + 1) -th precharge period. ) Is detected by the drain driver 73. The drain driver 73 outputs the voltages of the drain lines 43, 43,.

  Then, the voltage of the drain electrode 28 of the light receiving elements 20, 20,. The output is quantized by the A / D converter 87 and input to the controller 84. Specifically, a voltage (voltage drop) obtained by subtracting a voltage value reduced in accordance with the amount of incident light from a voltage value of the drain line 43 that has been raised by the precharge pulse is used as an output value of 256 gradation digital values. The gradation of the output value increases as the amount of incident light increases. The controller 84 records the output value of the A / D converter 87 in the memory 86 so that the output value distribution along the light receiving surface 35 of the solid-state imaging device 10 is two-dimensional image data (a set of output values of each pixel). Body) is recorded in the memory 86.

  Thereafter, the controller 84 operates the top gate driver 71, the bottom gate driver 72, and the drain driver 73 in a light sensing time of 3 seconds with the excitation light irradiation device 81 turned on.

In the memory 86, image data with a light sensing time of 3 seconds is recorded in addition to the light sensing time of 1 second.

Next, the controller 84 reads the first and second image data recorded in the memory 86.
Next, the controller 84 triples the output value of the pixel at the address of the first row and the first column of the first image data and the pixel at the address of the first row and the first column of the second image data. A weighted average obtained by dividing the sum of the output value and 4 is calculated and stored in the memory 86. Similarly, the controller 84 calculates a weighted average of all remaining pixels and stores it in the memory 86. Here, the pixel is composed of pixels of n rows and m columns.

Next, the controller 84 reads the slope A and the intercept B recorded in the storage device 82, and substitutes the obtained weighted average of each pixel into the output value of Expression (2) to obtain the incident light amount.
Next, the controller 84 records image data including the obtained address of each pixel and the amount of incident light in the memory 86.
Next, the controller 84 causes the output device 85 to output image data including the obtained gradation of each pixel. As a result, the output device 85 outputs an image. The molecular weight of the fluorescently labeled DNA can be calculated from the image data (the incident light quantity value) output by the output device 85, the standard incident light quantity value, and the standard molecular weight.

  According to the present embodiment, since it is used that the solid-state imaging device 10 has output characteristics as shown in FIG. Therefore, the dynamic range of the solid-state imaging device 10 is widened.

  Further, since the storage device 82 stores not only the database but the inclination A and the intercept B, the storage capacity of the storage device 82 can be minimized. Since the storage capacity of the storage device 82 is small, the manufacturing cost of the biopolymer detection device 80 and the biopolymer detection chip 1 can be reduced.

  Further, since the controller 84 calculates the correlation between the incident light quantity and the gradation value so as to be a linear function, it is not necessary to make the A / D converter 87 highly accurate, and scanning for scanning the optical system. No mechanism is required. Therefore, the biopolymer detection device 80 can be downsized, and the manufacturing cost of the biopolymer detection device 80 can be suppressed.

The present invention is not limited to the above-described embodiment, and various improvements and design changes may be made without departing from the spirit of the present invention.
In the above embodiment, the solid-state imaging device 10 using double gate transistors as the light receiving elements 20, 20,... Has been described as an example, but the present invention is applied to a solid-state imaging device using photodiodes as photoelectric conversion elements. Also good. Solid-state imaging devices using photodiodes include CCD image sensors and CMOS image sensors. In a CCD image sensor, photodiodes are arranged in a matrix on a substrate, and around each photodiode, a vertical CCD and a horizontal CCD for transferring an electrical signal photoelectrically converted by the photodiode. Is formed. In a CMOS image sensor, photodiodes are arranged in a matrix on a substrate, and a pixel circuit for amplifying an electrical signal photoelectrically converted by the photodiodes is provided around each photodiode. Yes. When the solid-state imaging device 10 is a CCD image sensor or a CMOS image sensor, it is a matter of course that the circuit configuration of the drive circuit 70 is changed to one suitable for the CCD image sensor or the CMOS image sensor. The drive circuits for CCD image sensors and CMOS image sensors are well known.
In the above embodiment, the drive circuit 70 is caused to perform the drive operation once with the optical sensing time set to 1 second, and the drive circuit 70 is driven with the optical sensing time set to 3 seconds. The drive circuit is driven three times with the optical sensing time set to 1 second, and the drive circuit 70 is driven with the optical sensing time set to 3 seconds. The action may be performed once and these sums may be divided by four.
In each of the above embodiments, the optical sensing time is 1 second and 3 seconds. However, the optical sensing time is not limited to this as long as it is arbitrarily different, and may be obtained by three or more types of optical sensing times.
In each of the above embodiments, each pixel is subjected to optical sensing a plurality of times in different seconds. For example, among a plurality of light receiving elements 20 corresponding to one spot 60, for example, one second in a specific row (for example, an odd row). The optical sensing is performed with the optical sensing time of 3 seconds, and the optical sensing is performed for another specific row (for example, even rows) with the optical sensing time of 3 seconds, and the weighted average and the incident light amount of the plurality of pixels of one spot 60 are obtained. May be. In this case, first, the driving operation for the light sensing time of 1 second is sequentially performed only for the odd-numbered pixels, and then the driving operation for the light sensing time of 3 seconds is sequentially performed only for the pixels of the even-numbered row.

It is the perspective view which showed the principal part of the biopolymer detection apparatus in embodiment of this invention. It is the top view which showed a part of biopolymer detection chip | tip schematically. It is the top view which showed the pixel of the solid-state imaging device. It is IV-IV arrow sectional drawing. It is the graph which showed the characteristic of the solid-state imaging device. It is the graph which showed the incident light quantity-output characteristic of the solid-state imaging device. It is the table | surface which showed the incident light quantity-output characteristic of the solid-state imaging device. It is drawing which showed the solid-state imaging device and its peripheral circuit. It is drawing which showed the solid-state imaging device and its peripheral circuit. It is the chart which showed transition of the signal of a solid imaging device. It is the block diagram which showed schematically the structure of the biophotopolymer detection apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Solid-state imaging device 20 Light receiving element 60 Spot 70 Drive circuit 80 Biopolymer detection apparatus 82 Memory | storage device 84 Controller

Claims (6)

A solid-state imaging device having a light receiving surface and a plurality of light receiving elements arranged below the light receiving surface;
A plurality of types of spots made of known biopolymers and scattered on the light receiving surface of the solid-state imaging device,
A driving circuit capable of outputting an output value corresponding to the amount of incident light of the light receiving element during the optical sensing time in which photoelectric conversion is performed by the light receiving element by driving the solid-state imaging device, and changing the light sensing time; ,
A controller that obtains a weighted average of the output value of the light receiving element when the optical sensing time is the first time and the output value of the light receiving element when the optical sensing time is the second time. A biopolymer detection device.   The biopolymer detection apparatus according to claim 1, wherein the controller sets a deviation rate between the weighted average and the linear approximation obtained from the weighted average to −5% to 5%.   The biopolymer detection apparatus according to claim 1, wherein the controller obtains a linear approximation regression equation from the weighted average.   The biopolymer detection apparatus according to claim 3, wherein the controller obtains an incident light amount of the light receiving element based on a linear approximation regression equation obtained from the weighted average.   The biopolymer detection apparatus according to claim 3, further comprising a storage device that stores a slope and an intercept of a regression equation that is linearly approximated from the weighted average.   6. The biopolymer detection apparatus according to claim 5, wherein an inclination and an intercept are read from the storage device, and an incident light amount value is obtained from the weighted average value at the time of measurement.

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