JP4099540B2 - Biological sample analysis chip and analysis method - Google Patents

Description

  The present invention relates to a probe chip for testing various test items at once, that is, a multi-item sensor. The target is DNA, RNA, and protein, and in particular, biological samples such as DNA chips for DNA testing that have recently attracted attention. Concerning analysis chip.

  Along with the progress of the genome project, the movement to understand living organisms at the DNA level, to diagnose diseases and to understand life phenomena has become active. It is effective to investigate the expression status of genes in order to understand life phenomena and investigate the functions of genes. As an effective method, a DNA probe array or a DNA chip in which a large number of DNA probes are classified and fixed on a solid surface (called an oligo chip because it is actually an oligonucleotide derivative) Is also used. Or recently, protein chips fixed in an array as various proteins (generally antibodies as probes) have been used.

  In order to make a DNA chip, a method of synthesizing oligomers of sequences designed in a large number of compartmented cells using photochemical reaction and lithography often used in the semiconductor industry one by one (Non-patent Document 1: Science 251, 767-773 (1991)), or a method of implanting DNA probes and protein probes one by one in each compartment (Non-Patent Document 2: Proc. Natl. Acad. Sci. USA 93, 4613-4918 (1996)) and so on.

  Each of these chips has a structure in which a large number of probes such as a slide glass are arranged in a plane by dividing a plurality of probes into sections. In general, which probe is located at which position is indexed only at a physical position where the probe is fixed.

  The method of use is to introduce a fluorescent substance that is introduced onto a substrate by hybridizing a sample polynucleotide (hereinafter simply referred to as a sample polynucleotide) such as a fluorescently labeled DNA fragment or mRNA to a probe on a chip substrate, or cDNA reversely transcribed from this Is detected with a fluorescence scanner. Alternatively, after hybridizing the sample polynucleotide, a fluorescently labeled oligo complementary to the sample polynucleotide adjacent to the probe is linked by ligation, or a fluorescently labeled dNTP substrate is reacted using DNA polymerase. Thus, it is the mainstream to detect the phosphor to be introduced onto the substrate.

  Recently, an electrochemical detection method using a redox reaction has been put into practical use. In the case of protein, using an affinity reaction such as an antigen-antibody reaction to capture a specific protein on the substrate and then analyzing with a mass spectrometer, a sandwich reaction is performed with a fluorescently labeled antibody or enzyme labeled antibody, and the substrate There are a method for detecting the phosphor and enzyme activity remaining above, and a detection method using electrochemiluminescence.

  In the electrochemiluminescence method, an antigen capturing antibody is present on the electrode surface. A ruthenium complex is used as a label for the sandwich antibody (Non-patent Document 3: Clin. Chem., 37, 1534-1539 (19991)). Ruthenium is oxidized on the electrode surface, and emits light when the ruthenium electrons, which are excited when they are reduced by being coupled with the redox reaction of TPA, fall to the ground state.

  As a detection method aiming at high-sensitivity and quantitative detection, an immunoassay is used in which a normal microscopic detection particle of about 700 μm is used as a label, and the number of reacted particles is counted with a counter to quantitatively detect the target substance. (Non-Patent Document 4: Anal. Biochem. 202, 120-125 (1992)).

Science 251, 767-773 (1991) Proc. Natl. Acad. Sci. USA 93, 4613-4918 (1996) Clin. Chem., 37, 1534-1539 (19991) Anal. Biochem. 202, 120-125 (1992))

  The current chip technology, particularly a DNA probe array or a DNA chip, uses fluorescence detection, so there is a limit to the size of an area for fixing a probe (hereinafter, an area for fixing one probe is defined as an element). That is, as long as light is used, the resolution generally has a limit (resolution) expressed by the following equation (1).

ここで媒質が空気なら開口数は０．８程度が限界である。したがって５３０ｎｍの光源を使用すると識別サイズとして４００ｎｍくらいが光を用いる検出限界となる。もちろん、蛍光検出であるから、蛍光強度が十分あれば、点光源としてより狭い範囲から発する光を検出することはできるし、ニアフィールド顕微鏡のように特殊な技術を用いればより狭い領域での光検出が可能になる。

If the medium is air, the numerical aperture is about 0.8. Accordingly, when a light source of 530 nm is used, the detection limit using light is about 400 nm as the identification size. Of course, because it is fluorescence detection, if the fluorescence intensity is sufficient, it is possible to detect light emitted from a narrower range as a point light source. Detection is possible.

  However, if it is attempted to detect fluorescence from such a narrow range, there is a problem that it is difficult to ensure sensitivity, for example, it is not possible to obtain a sufficient amount of light. In addition, even if the probe is fixed at a predetermined position, if the element becomes small, it is necessary to rely only on position information for indexing which element is being detected. It becomes difficult to separate from the element. For example, assuming that there is one million elements, and assuming an extreme state in which the sample polynucleotide is hybridized to only one of these elements, it is substantially difficult to identify the elements only with the position information where the probe is spotted in advance. Become.

  For this reason, in the prior art, the element position accuracy is 1 μm, the element size is 5 μm × 5 μm, the gap between the elements is 5 μm, and the number of elements is almost limited to about 1 million elements / chip. This is also the limit of lithography in which a DNA probe, which is a current polymeric biological material, is formed on a chip. Practically worse than this, the actual size of one element is about 30 μm × 30 μm.

  Furthermore, when using light, the restrictions in the case of multiple labeling are large due to the relationship between the excitation light source and the fluorescence wavelength. In general, even when coherent light is used as an excitation light source, the obtained fluorescence wavelength has a wavelength dispersion of several tens of nm. For this reason, when a single excitation wavelength is used, it is the limit to measure fluorescence by exciting about four types of phosphors. Even if a plurality of excitation wavelengths are used, only about 6 types of phosphors can be used in a practical range of 500 to 700 nm. For this reason, when detecting a plurality of target polynucleotides in the same element by the fluorescence method, about 4 types are considered to be the limit.

  Since DNA chips (DNA microarrays) are not quantitative in the first place, there are cases where a technique called differential hybridization or competitive hybridization is generally used. In this case, when there are two systems of samples A and B, phosphors having different wavelengths are labeled on each of A and B, and both samples are mixed. When hybridizing to the same DNA chip competitively in a mixed state, the intensity ratio of the two types of phosphors is detected according to the amount ratio of the two. In this method, it is possible to obtain data for the time being even on a DNA chip having poor quantitativeness and poor reproducibility. There are usually many types of samples to be compared, but it is difficult to apply a larger number of samples to differential hybridization due to the above-described wavelength dispersion of excitation light and fluorescence.

  Furthermore, in general, in an analytical device, ensuring quantitativeness, dynamic range, and reproducibility are important items. For methods and devices that do not reach a certain standard, they are not practical. However, regarding the DNA chip, since it is difficult to obtain a mixed sample whose concentration can be defined if the number of elements is too large, almost no study has been made.

  In order to solve these problems, an object of the present invention is to propose a polynucleotide chip and a protein chip with higher density and better quantitativeness and reproducibility. The chip shape, the chemistry used for the chip, and the detection method are proposed.

  In the present invention, an atomic force microscope technique that detects the shape of a substance by tracing atomic force is used for detection instead of light detection having a limit in resolution. In general, the atomic force microscope has a resolution capable of discriminating DNA molecules in a single strand and double strand (diameter of about 3 nm). However, in order to enable high-speed scanning, more easily detected nanoparticles can be detected. Detection is performed using as a label. In order to reliably index the elements, gaps are provided between the elements to isolate the elements from each other, and identification marks having different shapes are provided between the elements. Since nanoparticles can be produced with a particle size distribution of 10% in the range of about 5 to 40 nm, such nanoparticles are used as labels, and a group of particles having a particle size distribution of about 10 stages are simultaneously reacted and identified with an atomic force microscope.

According to the present invention, during the scanning with an atomic force microscope, it becomes possible to sequentially detect oligonucleotide fragments captured by a probe while identifying elements. Therefore, even if the elements are arranged at high density, the element can be reliably indexed and the sample target substance can be detected. For this reason, the size of one element can be about 700 nmφ or less, and a chip of about one million elements can be accommodated in about 1 mm ² . This is an element density exceeding the conventional method by two orders of magnitude in terms of area ratio.

Example 1
FIG. 1 is a conceptual view showing a part of the DNA chip of Example 1 of the present invention in a perspective view. The chip 100 is formed on a silicon substrate 101 having an oxide film surface. Each element 1 has a cylindrical shape of 700 nmφ. The interval 2 between the elements is 300 nm. That is, the elements are arranged at a pitch of 1 μm. The element is a unit of 50 × 50 and forms one element group as shown by being surrounded by a chain line 3, and each element group 3 is arranged in an array of 20 × 20. A groove 5 having a width of 40 nm and a depth of 20 nm exists between each element group 3, and indexing pillars 4 are provided at four corners of each element in the element group. The pillar 4 between adjacent elements is shared by each element. The pillar 4 has a cylindrical shape with a diameter of 17 steps at a minimum of 50 nmφ to 10 nm and a height of 17 steps every 5 nm to 10 nm, and the pillars 4 at the four corners of each element are used for indexing like a barcode. Therefore, the same pillar 4 appears nine times in one element group 3, but the pillars 4 of the same size are arranged so as to be as random as possible so that the pillars 4 of the same size do not appear continuously.

  FIG. 2 shows a more detailed relationship between each element 1 and the pillar 4 on the substrate 101 described in FIG. 1, and DNA probes 21, 22,. It is a figure which shows typically the relationship between DNA fragment 201,202,203 and the AFM probe 60 which detects this. Each element 1 on the substrate 101 has a structure that is one step higher than the surface of the substrate 101. That is, the elements are depressed like a groove and form a boundary between the elements. Element 1 has a height of 20 nm. The DNA probe and the like will be described later.

  The pillars 4 provided at the four corners of each element are used not only for the purpose of indexing but also for increasing the speed of probe hybridization. 3A, 3B, and 3C are overall views for explaining the effect of increasing the probe hybridization speed of the pillar 4, and FIG. 4 is a detailed explanatory view of the effect of FIG.

  As shown in FIG. 3A, an upper surface plate 102 capable of reciprocating movement is installed on the upper surface of the element 1 of the substrate 101 of the DNA chip 100 as indicated by an arrow 103 with a gap of 100 μm. 1 μl of the sample solution is sandwiched between the substrate 101 and the substrate 101. As shown in FIG. 3B, the top plate 102 is moved in the direction of the arrow with respect to the substrate 101. Next, as shown in FIG. 3C, the top plate 102 is moved with respect to the substrate 101 in the direction of the opposite arrow. The reciprocating motion of the top plate 102 is performed at 1 Hz. In general, in such a micro device, the liquid tends to be laminar and the stirring efficiency is lowered. However, by reciprocating the plate 102 on the upper surface of the substrate 101, the laminar flow is disturbed and hybridization is promoted. FIG. 4 is a cross section seen at the center position of the adjacent element 1, and shows a state where the pillar 4 is visible beyond the center position of the element 1. When the upper surface plate 102 is reciprocated with respect to the substrate 101 as indicated by the arrow 103, the movement of the liquid in the planar direction is forced according to the reciprocating motion as indicated by 105 and 106, so that the hybridization is a solute (here, It has been found that only diffusion in the thickness direction of a DNA sample or a labeled probe is rate-limiting (for example, JP-A-2004-144521). Therefore, the sample solution is always replaced on the surface of each element 1, and the reaction is promoted. Further, in the present invention, since substantially random pillars 4 stand between the elements 1, the liquid forcedly moved in the plane direction is more effectively stirred by the obstacle pillars 4 and further promotes the reaction. Is done. For this reason, the speed of hybridization approaches the reaction speed in the substantial liquid layer.

  Referring to FIG. 2 again, different DNA probes 21, 22,... Are fixed to the upper surface of each element 1 having a cylindrical structure of 700 nmφ and 20 nm in height for each element. The DNA probe used here is PNA. Since PNA does not have a negative charge derived from a phosphodiester bond unlike ordinary DNA, an electrostatic repulsive force does not act on the target DNA. This has the effect of increasing the efficiency of hybridization. Especially when a normal DNA probe is used in the case where the probe is fixed at a high density on the solid surface of the element like a DNA chip, the target DNA needs to approach the probe beyond the negatively charged barrier, Both reaction kinetics and thermodynamics are disadvantageous. The sample DNA must be single-stranded. By using a probe having no negative charge such as PNA, the charge on the element surface can be eliminated, so that the hybridization speed and yield are improved. Furthermore, since the characteristic of PNA having no charge does not generate an electrostatic repulsive force, even if the target DNA is double-stranded, it can be competitively pulled into the double-stranded and hybridize competitively.

In Example 1, each element 1 is immobilized with a DNA probe 21, 22,... Having a length of 45 to 60 bases in the region spanning the first and second exons of human cDNA. ing. This prevents false positives and a decrease in hybridization site due to hybridization of the remaining genome. The probe immobilization method is performed by a silane coupling reaction according to a conventional method. With respect to the implementation conditions, that is, the buffer solution composition and the sample DNA probe, the hybridization method described in Nucleic Acids Research (2002) 30, No. 16 e87 is followed. Regarding the probe fixing method, according to the above-mentioned literature, the silicon substrate 101 having an oxidized surface is treated with 3-aminopropyltrimethoxysilane to introduce amino groups on the surface. The amino group introduced on the surface and the SH group of the probe are cross-linked with N- (11-maleimidedecanoxyloxy) succinimide. When a probe of about 50 bases is immobilized by this method, the probe immobilization density is approximately 1 molecule per 15 nm ² . Thus, approximately 25,000 probe molecules are immobilized per element.

  As a sample, 1st strand cDNA obtained by reverse transcription of human leukocyte-derived mRNA once is used. In general, when taking full-length cDNA, the oligo cap method is used, but here we want to know the amount of mRNA as it is in the cell. Suspend dropwise in phenol with sonic agitation and instantly destroy cells. Purify total RNA according to standard methods. A reverse transcriptase is made to act on RNA contained in approximately 10 cells to obtain 1st strand cDNA.

The 1st strand cDNA thus obtained is dissolved in 1 μl of 2 × SSC and dropped onto the DNA chip. In this state, Leer of 1st strand cDNA is 10 ² molecule about, because it contains approximately 10 ⁵ molecules ones Abundant, the probe number and order basis of chip surface matching.

  Stir at 45 ° C. for 1 hour according to the method shown in FIGS. 3 and 4 to complete the hybridization. Thereafter, the top plate 102 for stirring described in FIGS. 3 and 4 is removed and dried. In Example 1, since a PNA probe is used, it is not always necessary to use a high salt concentration condition as in normal DNA chip reaction conditions. This is because it is not necessary to shield the negative charges of the DNA probe and sample DNA due to salt. Rather, lower ionic strength gives better results.

  In Example 1, 1st strand cDNA in a single-stranded state is used, but hybridization can be performed even with double-stranded DNA. In this case, if the ionic strength is not increased much, the sample double-stranded DNA itself is more easily dissociated by the repulsive force due to the negative charge of the sample, and the probe does not have a negative charge, so that it can hybridize without any problem. However, since this is basically a competition reaction between double-strand DNA stranding and probe hybridization, the yield of hybridization is lower than that of 1st strand cDNA hybridization.

  In Example 1, hybridization is completed at high speed even on the solid surface by using a PNA probe and sufficiently stirring. When a conventional DNA probe, which is a conventional technique, is used, mainly due to electrostatic repulsion, even if the sample concentration and the amount of probe described above are reacted for 24 hours, the reaction efficiency is poor and the reaction is not completed.

  FIG. 2 shows a state in which the sample DNA fragment 201 is hybridized to the probe 21 on the element 1.

  Next, in order to identify different sequence parts complementary to the DNA fragments of the sample DNA fragments 201, 202, 203 hybridized to the probe 21, gold nanoparticles 23 ′ hybridized to the sample DNA fragments 201, 202, 203, A second probe labeled 24 ', 25' is allowed. An exon different from the exon used for the capture probe 21 on the element 1 is used as the second probe at this time. For example, PNA probes corresponding to the sequences of exon 3, exon 4 and exon 5 are prepared. Each probe length consists of 30-50 bases. Each probe has a sulfid group (SH group) introduced at the 5 'end during synthesis. Gold nanoparticles of 8.3 nm, 11 nm, and 17 nm are mixed corresponding to the probes of exon 3, exon 4, and exon 5, respectively, and PNA probes labeled with gold nanoparticles having different particle sizes are obtained. The above three kinds of gold nanoparticle labeled probes (23 ′, 24 ′, 25 ′) are mixed at a concentration of 10 pmol / μl, dropped onto the DNA chip hybridized with the sample DNA, and again the method of FIGS. For 15 minutes.

  After washing with 2 × SSC, the top plate 102 for stirring described in FIGS. 3 and 4 is removed and dried. Thereafter, the surface of the chip 100 is two-dimensionally scanned while tapping the AFM probe 60 at 10 kHz as indicated by an arrow 61.

  FIG. 5 is a diagram schematically showing a position signal of the AFM probe 60 obtained by scanning the chip 100 shown in FIG. 1 with the AFM probe 60 in the lateral direction. From the position signal of the AFM probe 60, it can be seen that the element 1 is located at the broken line 41, and it can be seen that the gap 2 between the elements 1 corresponds to the region 45 adjacent thereto. It can also be seen that pillars of different sizes are shown by broken lines 51 and 52. Signals at positions denoted by reference numerals 47, 48, and 49 are sent to the PNA probe fixed on the element 1 and the second probes (23 ′, 24 ′, FIG. 25 ′) is a signal indicating that a PNA probe hybridized and hybridized with a gold nanoparticle is hybridized thereto. Therefore, the position of the element and the label can be recognized from the position signal of the AFM probe 60.

(Example 2)
FIGS. 6A, 6B, and 6C are overall views for explaining another example of the effect of increasing the probe hybridization speed of the pillar 4, and FIG. 7 is a detailed explanatory view of the effect of FIG. It is.

  As is clear by comparing FIGS. 6A, 6B, and 6C with FIGS. 3A, 3B, and 3C, in Example 2, on the substrate 101 of the DNA chip 100, FIG. The only difference is that a rotating rod 120 having a diameter of 100 μm is provided on the upper surface instead of the upper surface plate 102. The rod 120 can rotate at 200 rpm while being in contact with the sample solution 122 on the substrate 101. The gap between the rod 120 and the chip element is 100 μm. The rotation of the rod is a spindle motor, and the movement to the left and right is to move the chip on the stage to the left and right at a rate of once every 10 seconds. 6 μl of sample solution or nanoparticle-labeled probe solution is dropped onto the substrate 101 and moved from left to right as shown in FIGS. 6A, 6B, and 6C while the rod 120 is rotated. Further, if necessary, it is moved from right to left (arrow 129). By rotating the rod 120, the movement of the liquid in the planar direction is forced as shown by the thick lines 115 and 125, so that the solute is sufficiently stirred on the substrate 101. Therefore, the sample solution is always replaced on the surface of each element 1, and hybridization is promoted. Further, in the second embodiment, since the pillars 4 having substantially random sizes stand between the elements 1, the solution forcedly moved in the plane direction is disturbed in the liquid flow by the pillars 4 that are obstacles. The reaction is further promoted. In Example 2, the reaction solution for hybridization is sufficiently stirred on the element of the chip by the interaction between the rotation and movement of the rod 120.

  The size of the chip of the present invention will be described. In general, when an object is to be recognized using light, an object having a wavelength of 400 to 500 nm or less cannot be detected in the above-described formula (1) and the numerical aperture of 0.8. Below this, even objects cannot be recognized. In practice, the limit of about 700 nm becomes a limit when trying to define the life of an object.

  In the present invention, since the substance is detected by measuring the atomic force, the substance can be measured without depending on the wavelength. For this reason, even if the element 1 or the element interval is 700 nm or less, the separation between the element 1 itself and the element 1 can be detected. In the first embodiment, the element 1 has a size of 700 nmφ and the element interval is 300 nm. However, the element 1 may have a finer structure or the element interval. For example, even when the element 1 is 500 nm or less or the element interval is 300 nm or less, 8.3 nm gold nanoparticles captured by the hybridization reaction on the element 1 can be quantitatively detected with an atomic force microscope.

Considering the lower limit of element 1 and the element interval, 70 nm is the lower limit for the following reason. That is, as described above, when a probe of about 50 bases is immobilized, the probe immobilization density is approximately 1 molecule per 15 nm ² . On the other hand, the lower limit of the size of the nanoparticles used for detection is 5 nm, which is generally available in the current state of the art, so it is almost equal to the probe fixing density, and this is one criterion for representing the lower limit. Actually, if there are not at least 1000 particles on the element 1, there is a problem from the viewpoint of quantitative detection. That is, the size lower limit of the element 1 is 15 × 1000 nm ² . Therefore, the lower diameter limit of the size of the circular element 1 is 70 nm. Of course, if quantitativeness is not a problem, smaller elements can be used. In this case, the region where one probe molecule is closed when the probe is fixed becomes a limit. That is, 3 nm is the minimum unit of the element.

(Example 3)
A protein chip is obtained in which affinity purified polyclonal anti-AFP antibody and anti-CEA antibody are immobilized on different elements. Separately, an SH group is introduced into an F (ab ′) ₂ fragment obtained by papain degradation of the same anti-AFP antibody and anti-CEA antibody. The number of SH groups to be inserted is 3 to 4 molecules per F (ab ′) ₂ molecule. Mix with 20 nm gold nanoparticles to obtain particles with F (ab ′) ₂ bonded to the surface. As a sample, prepare AFP alone at a concentration of 1 zmol / μl, CEA alone at a concentration of 5 zmol / μl, and a control containing nothing. The solvent is PBS (pH 7.4) containing 0.1% Tween 20 and 0.5% BAS. Each solution is reacted with a protein chip, and further, gold nanoparticle label F (ab ′) ₂ is reacted. The reaction time is 5 minutes for the first sample reaction and 5 minutes for the reaction of the gold nanoparticle labeled F (ab ′) ₂ . After completion of the reaction, the reaction mixture is washed with a buffer containing 0.1% Tween 20 and 0.5% BAS and detected by AFM.

  In the chip reacted with AFP, 120 particles / element are detected in the element to which the anti-AFP antibody is bound, and 6 particles / element are detected in the element to which the anti-CEA antibody is immobilized. In the chip reacted with CEA, 7 particles / element are detected in the element to which the anti-AFP antibody is bound, and 1340 particles / element are detected in the element to which the anti-CEA antibody is immobilized.

  In the control solution, only 2-4 particles / element of gold nanoparticles are detected in any element on the chip.

  As described above, the present invention can be implemented in the form of several examples. In any case, the hybridized sample DNA and the like are detected substantially every molecule, so that the sensitivity can be increased. Can achieve sensitivity exceeding conventional methods. Since it is possible to detect a very small amount of DNA (RNA) in a very small volume, it becomes possible to detect a target DNA (RNA) without pretreatment amplification by PCR, which was impossible in the past. Since the size and shape of the labeled particles can be changed for detection, it is possible to perform multi-analysis of 6 to 10 different samples with the same element. In addition to the use for conventional differential hybridization, this technique enables the sample polynucleotide to be captured on the element with the same probe and detected with a different labeled probe. The multi-analysis technique of the present invention has an advantage that alternative splicing detection and multiple SNP typing can be performed with one element.

  FIG. 8 is a perspective view showing an example of the configuration of an AFM cantilever suitable for implementing the present invention. As shown in FIG. 8, the AFM cantilever 130 may have a structure in which a plurality of needles 131 to 134 are arranged in an array. The needles 131 to 134 are independently attached to the levers 141 to 144 to which the piezoelectric elements 151 to 154 are attached, and have a structure in which the electromotive force of the piezoelectric elements changes and can be detected corresponding to the upper and lower sides of the needle. By using such an array-shaped cantilever, a plurality of lines can be scanned together. For this reason, it is possible to train nanoparticles captured on the chip at high speed. Furthermore, by taking advantage of the capability of high-speed scanning, the shape trace error can be reduced by scanning the same portion multiple times. Specifically, by performing scanning N times, the measurement error in the height direction can be reduced to 1 / √N. In the embodiment of this specification, ten cantilevers are used, and the value in the depth direction is calculated by repeating the scan 16 times. Thereby, a 1 mm square chip composed of 10,000 elements can be scanned in 30 minutes.

The conceptual diagram which shows a part of DNA chip of Example 1 of this invention with a perspective view. A more detailed relationship between each element 1 on the substrate 101 described in FIG. 1 and the pillar 4, DNA probes 21, 22,... Fixed on each element 1, and a DNA fragment 201 hybridized thereto. And a diagram schematically showing the relationship between the AFM probe 60 that detects this. (A), (b), (c) is the whole figure explaining the effect which speeds up the probe hybridization speed of the pillar 4. FIG. 4 is a detailed explanatory diagram of the effect of FIG. 3. FIG. 3 is a diagram schematically showing a position signal of the AFM probe 60 obtained while scanning the chip 100 shown in FIG. 1 with the AFM probe 60 in the lateral direction. (A), (b), (c) is the whole figure explaining the effect which speeds up the probe hybridization speed | rate of the pillar 4 of Example 2. FIG. FIG. 7 is a detailed explanatory diagram of the effect of FIG. 6. The perspective view which shows the example of a structure of the AFM cantilever suitable for implementation of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Element, 2 ... Element interval, 3 ... Element group, 4 ... Pillar, 5 ... Groove, 6 ... 21, 22 ... DNA probe, 23 ', 24', 25 '... Gold nanoparticle, 50 ... Cell separation Chip, 51 ... sample introduction part, 60 ... AFM probe, 100 ... chip, 101 ... silicon substrate, 102 ... top plate, 121 ... rod, 201 ... DNA fragment, 130 ... AFM cantilever, 131-134 ... needle, 141- 144: Lever, 151-154: Piezoelectric element.

Claims (6)

A biological sample analysis chip in which a plurality of independent regions are provided on a substrate, and different probes are fixed to the respective regions, wherein each of the independent regions has a circular area of 700 nmφ or less. state, and are circular area over 3Nmfai, with arranging a structure with a specific shape the four corners of the plurality of areas in which the independent, said structure having a specific shape disposed on these four corners A biological sample analysis chip characterized by being different for each of a plurality of independent regions . Wherein a plurality of independent regions of area in units of predetermined number, claim 1 Symbol placing of the biological sample analysis chip are arranged with a groove formed between the regions. Wherein a plurality of independent regions of area in units of predetermined number, claim 1 Symbol placing of the biological sample analysis chip are spaced apart a predetermined distance or more. In the biological sample analysis chip in which a plurality of independent regions are provided on the substrate and different probes are fixed to the respective regions, the individual sizes of the plurality of independent regions are less than a circular area of 700 nmφ, 3 nmφ circular der more area is, with arranging a structure with a specific shape the four corners of the plurality of areas in which the independent, a structure with a specific shape disposed on these four corners were the independent A sample solution is dropped on a biological sample analysis chip that is different for each of a plurality of regions, and a thin plate or a rod that rotates at a predetermined speed is disposed on the top surface of the chip, and this is moved left and right on the substrate. An analysis method using a biological sample analysis chip, which promotes hybridization between a probe and a sample in a sample solution. In the biological sample analysis chip in which a plurality of independent regions are provided on the substrate and different probes are fixed to the respective regions, the individual sizes of the plurality of independent regions are less than a circular area of 700 nmφ, 3 nmφ circular der more area is, with arranging a structure with a specific shape the four corners of the plurality of areas in which the independent, a structure with a specific shape disposed on these four corners were the independent A biological sample analysis method for detecting a sample hybridized to a probe by tracing a biological sample analysis chip that is different for each of a plurality of regions with an atomic force microscope probe. 6. The method of analyzing a biological sample according to claim 5 , wherein the sample hybridized with the probe is reacted with a labeled probe labeled with particles of different particle diameters with an oligo probe complementary to the hybridized DNA fragment and hybridized with a different sequence portion. .

Images