公开(公告)号：WO1998027224A1

公开(公告)日：1998-06-25

申请号：PCT/JP1997004631

申请日：1997-12-16

Applicant: LABORATORY OF MOLECULAR BIOPHOTONICS ,  SHIONO, Hirofumi ,  NOHTA, Hitoshi ,  UTSUYAMA, Chika

Inventor： LABORATORY OF MOLECULAR BIOPHOTONICS

IPC: C12Q01/42

CPC classification number: C12Q1/34 ,  C12Q2334/00 ,  C12Q2334/20

Abstract: Enzyme substrates each carrying in its molecule a group which is cleaved by the enzymatic reaction and another group which is cleaved by the enzymatic reaction and then forms a coumarin derivative by intramolecular lactonization. A method for assaying enzymatic activity which comprises effecting an enzymatic reaction with the use of the above substrate and then measuring the fluorescence of the coumarin derivative thus formed to thereby determine the enzymatic activity.

Abstract translation: 各自携带分子中的酶底物是通过酶促反应切割的基团和另一个被酶反应切割的基团，然后通过分子内内酯形成香豆素衍生物。 用于测定酶活性的方法，其包括使用上述底物进行酶促反应，然后测量由此形成的香豆素衍生物的荧光从而确定酶活性。

公开(公告)号：WO1998011210A1

公开(公告)日：1998-03-19

申请号：PCT/JP1997003232

申请日：1997-09-12

Applicant: LABORATORY OF MOLECULAR BIOPHOTONICS ,  ABE, Satoshi ,  SATO, Yoshihiro

Inventor： LABORATORY OF MOLECULAR BIOPHOTONICS

IPC: C12N15/11

CPC classification number: C12Q1/6834 ,  C12Q2537/143 ,  C12Q2533/107 ,  C12Q2525/307

Abstract: A solid phase for target nucleic acid detection which has a base sequence hybridizable in sequence with a specified polynucleotide sequence of a target nucleic acid and contains a set of probes with their spatial arrangement immobilized onto the solid phase through a linker portion so as to be enzymatically ligated during the hybridization; and a method of target nucleic acid detection which comprises hybridizing the solid phase with the target nucleic acid, ligating the probes by a ligase reaction, and detecting the product of ligation.

Abstract translation: 用于靶核酸检测的固相，其具有与靶核酸的指定多核苷酸序列可序列杂交的碱基序列，并且含有一组其空间排列的探针通过接头部分固定在固相上以便酶促 在杂交期间连接; 和靶核酸检测方法，其包括将固相与靶核酸杂交，通过连接酶反应连接探针，并检测连接产物。

公开(公告)号：WO1997047968A1

公开(公告)日：1997-12-18

申请号：PCT/JP1997001960

申请日：1997-06-09

Applicant: LABORATORY OF MOLECULAR BIOPHOTONICS ,  HOSOI, Shigeru ,  KOJIMA, Makiko ,  KADOUCHI, Sachiko

Inventor： LABORATORY OF MOLECULAR BIOPHOTONICS

IPC: G01N33/533

CPC classification number: G01N33/582 ,  C12Q1/6834 ,  G01N33/533 ,  Y10S435/968 ,  Y10S436/828 ,  C12Q2563/107 ,  C12Q2545/114 ,  C12Q2565/601

Abstract: A fluorescent immunoassay characterized by labeling a sample with a fluorescent marker having a nucleic acid moiety stained with such a sufficient number of fluorochromes as to enable the measurement of fluorescent spots and a reactive group capable of binding specifically to the sample, immobilizing the sample thus labeled onto a solid phase, and then counting the fluorescent spots. The nucleic acid moiety of the fluorescent marker is a double- or single-stranded nucleic acid. Staining with the fluorochromes is effected with the use of intercalater type fluorochrome(s), mirror group type fluorochrome(s), and fluorochrome(s) covalently bonded to the nucleic acid.

Abstract translation: 荧光免疫测定法，其特征在于用荧光标记物标记样品，所述荧光标记具有用足够数量的荧光染料染色的核酸部分，以便能够测量荧光斑点和能够特异性结合样品的反应性基团，固定如此标记的样品 固相，然后计数荧光斑点。 荧光标记物的核酸部分是双链或单链核酸。 用荧光染料染色是通过使用与核酸共价结合的嵌入型荧光染料，镜式荧光染料和荧光染料来实现的。

公开(公告)号：WO1997047639A1

公开(公告)日：1997-12-18

申请号：PCT/JP1997001959

申请日：1997-06-09

Applicant: LABORATORY OF MOLECULAR BIOPHOTONICS ,  SHIONO, Hirofumi ,  KODAMA, Hirofumi ,  KOJIMA, Makiko

Inventor： LABORATORY OF MOLECULAR BIOPHOTONICS

IPC: C07H21/02

CPC classification number: C07H21/00

Abstract: A photocleavable cyclic oligonucleotide having a base sequence capable of hybridizing with a target DNA or RNA and a structure cyclized with a photocleavable linkage. The oligonucleotide is not decomposed by nucleases in the living body owing to its cyclic structure, so that it can be diffused to predetermined positions in the body by taking a sufficiently prolonged time. Further, the linkage is cleaved by irradiation with a suitable light after the lapse of a predetermined time, by which the cyclic oligonucleotide is converted into a linear one, thus acting as an antisense oligonucleotide.

Abstract translation: 具有能够与靶DNA或RNA杂交的碱基序列的可光切割的环状寡核苷酸和可光切割性连接环化的结构。 寡核苷酸由于其循环结构而不被生物体中的核酸酶分解，从而可以通过充分延长时间将其扩散到体内的预定位置。 此外，在经过预定时间之后，通过用合适的光照射使得连接被切割，由此将环状寡核苷酸转化为线性寡核苷酸，从而起到反义寡核苷酸的作用。

公开(公告)号：WO1997032198A1

公开(公告)日：1997-09-04

申请号：PCT/JP1997000513

申请日：1997-02-24

Applicant: LABORATORY OF MOLECULAR BIOPHOTONICS ,  OKAZAKI, Shigetoshi ,  MATSUMOTO, Hiroyuki

Inventor： LABORATORY OF MOLECULAR BIOPHOTONICS

IPC: G01N21/75

CPC classification number: G01N21/552

Abstract: A method and apparatus for determining enzyme activities by supplying a substrate solution and an enzyme solution to a reaction vessel to which a total reflection absorption prism is connected. An assay solution (40) comprising a mixture of an enzyme solution and a substrate solution is brought to a constant temperature in a reaction vessel (26) by a thermostat (23) and stirred by an agitator (21). An infrared ray (36) sent out of an infrared ray source (3) enters an interface between a total reflection absorption prism (28) in contact with the assay solution (40) and the solution (40) from the side of the prism (28), and is totally reflected thereupon. The spectrum of the totally reflected, outgoing transmitted infrared ray (37) is detected by an infrared ray detector (5), and the variation of the infrared ray absorption spectrum or absorbance is determined on the basis of what has been detected, whereby the enzyme reaction in the object solution (40) is assayed.

Abstract translation: 一种通过向连接有全反射吸收棱镜的反应容器提供底物溶液和酶溶液来测定酶活性的方法和装置。 将包含酶溶液和底物溶液的混合物的测定溶液（40）通过恒温器（23）在反应容器（26）中达到恒定温度，并通过搅拌器（21）搅拌。 从红外线源（3）发出的红外线（36）进入与测定溶液（40）接触的全反射吸收棱镜（28）和来自棱镜侧的溶液（40）之间的界面 28），并完全反映在那里。 通过红外线检测器（5）检测全反射出射透射红外线（37）的光谱，基于检测到的内容来确定红外线吸收光谱或吸光度的变化，由此酶 测定目标溶液（40）中的反应。

公开(公告)号：WO1998013676A1

公开(公告)日：1998-04-02

申请号：PCT/JP1997003391

申请日：1997-09-24

Applicant: LABORATORY OF MOLECULAR BIOPHOTONICS ,  TOYONAGA, Shuji ,  SHIROSHITA, Masahisa ,  SUGA, Takayuki ,  NAKANO, Yoshitaro

Inventor： LABORATORY OF MOLECULAR BIOPHOTONICS

IPC: G01J04/00

CPC classification number: G01N21/6445 ,  G01J3/4406 ,  G01J3/447 ,  G01J4/00 ,  G01N21/65

Abstract: The polarization of fluorescent light or Raman scattering light which is emitted from a sample when light is applied to the sample is measured with high accuracy. Excitation light which is emitted from a pulse excitation light source (1) and is p-polarized by a polarizer (2) and a half-wave plate (3) is applied to a sample (7) and the p-polarization component intensity Ipp and s-polarization component intensity Ips of the emitted fluorescent light are measured by photodetectors (13 and 14). In the same way, s-polarized excitation light is applied to the sample (7) and the p-polarization component intensity Isp and s-polarization component intensity Iss of emitted fluorescent light are measured. The G-factor is obtained from those measured values by a following formula: G = [(Ipp.Isp)/(Ips.Iss)] . Polarization responsiveness correction is performed in accordance with the G-factor to obtain the polarization of the fluorescent light.

Abstract translation: 以高精度测量向样品施加光时从样品发出的荧光或拉曼散射光的极化。 从脉冲激励光源（1）发射并由偏振器（2）和半波片（3）p极化的激发光施加到样品（7）上，并且p偏振分量强度Ipp 并且通过光电检测器（13和14）测量发射的荧光的s偏振分量强度Ips。 以相同的方式，对样品（7）施加s偏振激发光，并测量发射荧光的p偏振分量强度Isp和s偏振分量强度Iss。 G因子通过下式由这些测量值获得：G = [（Ipp.Isp）/（Ips.Iss）] 1/2。 根据G因子执行极化响应性校正，以获得荧光的偏振。

公开(公告)号：WO1998013524A1

公开(公告)日：1998-04-02

申请号：PCT/JP1997003438

申请日：1997-09-26

Applicant: LABORATORY OF MOLECULAR BIOPHOTONICS ,  SATO, Yoshihiro ,  TSUJI, Akihiko ,  SUGA, Takayuki

Inventor： LABORATORY OF MOLECULAR BIOPHOTONICS

IPC: C12Q01/68

CPC classification number: C12Q1/6818

Abstract: Detection probes enabling very convenient and highly accurate and sensitive detection of DNAs or RNAs having specific base sequences in specimens in the presence of the probes in excess of the target nucleic acid; and a detection method. When mixed with a specimen containing the target substance (DNA, RNA, etc.) having a polynucleotide having a specific base sequence, two types of fluorescent label detection probes hybridize, while being adjacent to each other, with the target nucleic acid. As a result, there arises a shift of the resonance energy between the two types of fluorescent dye molecules. When the resonance energy shift occurs in the hybrid thus formed, the fluorescence attenuation of the acceptor fluorescent dye molecule can be sufficiently delayed compared with the fluorescence attenuation of the directly excited acceptor by appropriately regulating (1) the number of bases between the two nucleotides to which the fluorescent dye moleculees have bonded; (2) the structure (double- or single-stranded) of the hybrid between the two nucleotides to which the fluorescent dye molecules have bonded; and (3), on the detection probes, the sites of the nucleotides into which the fluorescent dye molecules are to be introduced; and by (4) using a pair of detection probes with the appropriate selection of the fluorescent dye molecules. The detection method comprises using the above-mentioned detection probes and detecting the target substance by measuring changes in the fluorescence attenuation curve of the acceptor after the irradiation with pulse excitation rays, thus enabling highly accurate and sensitive detection of the target nucleic acid in the presence of the detection probes in excess of the target nucleic acid.

Abstract translation: 检测探针能够在超过目标核酸的探针存在下，在标本中具有特异性碱基序列的DNA或RNAs的非常方便和高度准确和灵敏的检测; 和检测方法。 当与含有具有特定碱基序列的多核苷酸的靶物质（DNA，RNA等）的样品混合时，两种类型的荧光标记检测探针在彼此相邻的情况下与靶核酸杂交。 结果，在两种类型的荧光染料分子之间产生共振能量的偏移。 当共轭能量偏移发生在如此形成的杂化物中时，与直接激发的受体的荧光衰减相比，受体荧光染料分子的荧光衰减可以通过适当地调节（1）两个核苷酸之间的碱基数 荧光染料分子结合; （2）荧光染料分子键合的两个核苷酸之间的杂交体的结构（双链或单链） 和（3），在检测探针上，将引入荧光染料分子的核苷酸位点; 并且通过（4）使用具有适当选择荧光染料分子的一对检测探针。 检测方法包括使用上述检测探针并通过测量在用脉冲激发射线照射之后受体的荧光衰减曲线的变化来检测目标物质，从而使得能够在存在下高度精确和灵敏地检测靶核酸 的检测探针超过目标核酸。

公开(公告)号：EP1016731A1

公开(公告)日：2000-07-05

申请号：EP99900152.2

申请日：1999-01-08

Applicant: Laboratory of Molecular Biophotonics

Inventor： ABE, Satoshi, Laboratory of Molecular Biophotonics ,  KODAMA, Hirofumi

IPC: C12Q1/68

CPC classification number: C12Q1/6862 ,  C12Q1/6823 ,  C12Q1/6834 ,  C12Q2565/543 ,  C12Q2521/501 ,  C12Q2523/107 ,  C12Q2521/101

Abstract: This invention relates to a pair of probes for the detection of a target nucleic acid, said pair of probes comprising (1) probe 1 having base sequence B1 complementary to A1 between two specific sequential base sequences A1 and A2 of the nucleic acid, wherein (a) the 5'-terminus of base sequence B1 is phosphorylated and (b) a first solid phase immobilizing part is bound to the 3'-terminus of base sequence B1, and (2) probe 2 having base sequence B2 complementary to A2 between base sequences A1 and A2, wherein (a) the 5'-terminus of base sequence B2 is bound to one end of a cleavage part and (b) a second solid phase immobilizing part is bound to the other part of the cleavage part. When the target nucleic acid is hybridized above the solid phase of the invention where said probes are immobilized on its surface, the probes on the solid phase occupy spatial positions beneficial to the formation of a hybrid and thus forms the hybrid efficiently. Therefore, probe 1 and probe 2 are efficiently ligated by ligase reaction. Furthermore, after the target nucleic acid is removed from the hybrid, only probe 2 ligated as described above will be able to exist on the solid phase through cleavage reaction of the cleavage part. Therefore, after the free probe 2 has been removed by washing, it will become possible to detect the presence of probe 2 on the solid phase ligated as described above, with high sensitivity and high recognition. This will enable detection of the presence of a target nucleic acid with higher sensitivity and higher recognition as compared to methods in the prior art.

Abstract translation: 本发明涉及到一对探针的用于检测靶核酸的，所述一对探针，其包括核酸，worin的两个特定顺序的碱基序列A1和A2之间的（1）探针具有1碱基序列A1互补B1的（ 一个）的碱基序列B1的5'末端被磷酸化和（b）第一固相固定部分结合到碱基序列B1的3'末端，和（2）样品2具有碱基序列B2互补A2之间 碱基序列A1和A2，worin的（a）碱基序列B2的5“末端结合到切割部分的一端和（b）第二固相固定部分结合到切割部分的另一部分。 当靶核酸，其中所述探针在其表面上固定化本发明的固相以上的杂交，在固相上探针占据的杂交体的形成有利的空间位置，从而有效地形成混合。 因此，探针1和探针2被有效地通过连接酶反应连接。 进一步，在靶后核酸从混合去除，只有两个测试连接如上所述将能够通过切割部分的切割反应在固相存在。 因此，免费试用2已经通过洗涤除去后，将成为能够检测上如上述那样，具有高灵敏度和高识别连接在固相样品2的存在。 这将使一个靶核酸与更高的灵敏度和更高的识别的存在的检测相比，在现有技术中的方法。

公开(公告)号：EP0892067A1

公开(公告)日：1999-01-20

申请号：EP97947958.1

申请日：1997-12-16

Applicant: Laboratory of Molecular Biophotonics

Inventor： SHIONO, Hirofumi, Lab. Molecular Biophotonics ,  NOHTA, Hitoshi, Lab. Molecular Biophotonics ,  UTSUYAMA, Chika, Lab. Molecular Biophotonics

IPC: C12Q1/42 ,  C12Q1/44 ,  C12Q1/34 ,  C12Q1/54

CPC classification number: C12Q1/34 ,  C12Q2334/00 ,  C12Q2334/20

Abstract: The enzyme substrate according to this invention has within its molecule both a group to be cleaved by an enzyme reaction and a group that forms a strongly fluorescent coumarin derivative through intramolecular lactonization when cleaved by the enzyme reaction. Furthermore, the method for determining an enzyme activity according to this invention comprises conducting the enzyme reaction by the use of the enzyme substrate of the invention and determining the enzyme activity by means of the measurement of fluorescence of the coumarin derivative formed.

Abstract translation: 根据本发明的酶底物在其分子内具有通过酶反应切割的基团和当通过酶反应切割时通过分子内内酯形成强荧光香豆素衍生物的基团。 此外，根据本发明的确定酶活性的方法包括通过使用本发明的酶底物进行酶反应，并通过测量形成的香豆素衍生物的荧光来测定酶活性。

公开(公告)号：EP0892067B1

公开(公告)日：2002-03-13

申请号：EP97947958.1

申请日：1997-12-16

Applicant: Laboratory of Molecular Biophotonics

Inventor： SHIONO, Hirofumi, Lab. Molecular Biophotonics ,  NOHTA, Hitoshi, Lab. Molecular Biophotonics ,  UTSUYAMA, Chika, Lab. Molecular Biophotonics

IPC: C12Q1/42 ,  C12Q1/44 ,  C12Q1/34 ,  C12Q1/54

CPC classification number: C12Q1/34 ,  C12Q2334/00 ,  C12Q2334/20