WO2019100841A1 - 检测核酸特异性和/或非特异性吸附的方法 - Google Patents

检测核酸特异性和/或非特异性吸附的方法 Download PDF

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WO2019100841A1
WO2019100841A1 PCT/CN2018/107248 CN2018107248W WO2019100841A1 WO 2019100841 A1 WO2019100841 A1 WO 2019100841A1 CN 2018107248 W CN2018107248 W CN 2018107248W WO 2019100841 A1 WO2019100841 A1 WO 2019100841A1
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image
detecting
substrate
probe
nucleic acid
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PCT/CN2018/107248
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English (en)
French (fr)
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赵陆洋
黄天逊
吴增丁
颜钦
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深圳市瀚海基因生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

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  • the invention relates to the technical field of nucleic acid detection, and in particular to a method for detecting nucleic acid specific and/or non-specific adsorption.
  • Non-specific adsorption affects the amount of valid data obtained and the accuracy of the test results.
  • Qualitative or quantitative detection of non-specific adsorption conditions can determine the quality of the chip, the surface modification of the chip, and the prediction of the test results.
  • Embodiments of the present invention are directed to at least one of the technical problems existing in the prior art or at least provide a useful commercial choice. To this end, the present invention provides a method of detecting nucleic acid specific and/or non-specific adsorption.
  • a method for detecting nucleic acid-specific and/or non-specific adsorption comprising: reacting a nucleic acid to be tested with a first probe, the first probe being immobilized on a surface of the first substrate, The nucleic acid to be tested is at least partially complementary to the first probe, the nucleic acid to be tested carries a first label, and the first probe carries a second label, the first label can generate a first signal, and the second label can generate a second signal; detecting a signal on the surface of the first substrate to obtain a first detection result; reacting the nucleic acid to be tested with a second probe, wherein the second probe is fixed on a surface of the second substrate, the second probe Carrying the second mark, the nucleic acid to be tested is not complementary to the second probe; detecting the signal on the surface of the second substrate to obtain a second detection result; detecting based on the first detection result and the second detection result Specific and/or non-specific ad
  • a method for detecting non-specific adsorption of a nucleic acid comprising: reacting a nucleic acid to be tested with a third probe, wherein the third probe is immobilized on a surface of the third substrate, the nucleic acid to be tested a third mark, the third probe has a fourth mark, the nucleic acid to be tested is not complementary to the third probe, the third mark can generate a third signal, and the fourth mark can generate a fourth signal; a signal on the surface of the third substrate, obtaining a third detection result; reacting the nucleic acid to be tested with the surface of the fourth substrate; detecting a signal on the surface of the fourth substrate to obtain a fourth detection result; based on the third detection result and the above Four test results, detecting non-specific adsorption of the above-mentioned nucleic acid to be tested.
  • a method for detecting nucleic acid-specific and/or non-specific adsorption comprising: reacting a nucleic acid to be tested with a fifth probe, the fifth probe being immobilized on a surface of the fifth substrate, to be tested
  • the nucleic acid is at least partially complementary to the fifth probe, the nucleic acid to be tested carries a fifth mark, the fifth probe carries a sixth mark, the fifth mark can generate a fifth signal, and the sixth mark can generate a sixth signal; Detecting a signal on the surface of the fifth substrate to obtain a fifth detection result; reacting the nucleic acid to be tested with a sixth probe, wherein the sixth probe is fixed on a surface of the sixth substrate, and the sixth probe is provided with the above a six-label, the nucleic acid to be tested is not complementary to the sixth probe; detecting a signal on the surface of the sixth substrate to obtain a sixth detection result; reacting the nucleic acid to be tested with the surface of the seventh
  • the above method for detecting non-specific adsorption of nucleic acid can qualitatively or quantitatively detect non-specific adsorption and/or specific adsorption of a target nucleic acid on a surface of a substrate and/or a surface of a substrate; and detecting a signal on a surface of the substrate by a detection system, Ability to qualitatively or quantitatively distinguish between specific and non-specific adsorption, to obtain information on the quantification and distribution of non-specific or specific adsorption, and to be used in chip production and in all methods or applications involving microarray detection of nucleic acid processes, such as for no-load Evaluation of the performance of the chip (without probe) or capture chip (with probe), quality control of chip production, prediction of the effect of chip capture nucleic acid, analysis and comparison.
  • FIG. 1 is a schematic flowchart diagram of an image processing method according to an embodiment of the present invention
  • FIG. 2 is another schematic flowchart of an image processing method according to an embodiment of the present invention.
  • FIG. 3 is a schematic view showing the principle of hybridization of a probe and a nucleic acid to be tested on a surface of a substrate according to an embodiment of the present invention
  • FIG. 4 is a schematic view showing the principle of hybridization of a probe and a nucleic acid to be tested on the surface of another substrate in an embodiment of the present invention
  • 5 is a schematic diagram showing the principle of hybridization of nucleic acid to be tested on the surface of another substrate in an embodiment of the present invention
  • FIG. 6 is an image of a Cy3 fluorescent dot (left image) and a Cy5 fluorescent dot (right image) in a field of view obtained by photographing an experimental group in an embodiment of the present invention
  • FIG. 7 is an image of a Cy3 fluorescent dot (left image) and a Cy5 fluorescent dot (right image) in a field of view obtained by taking a control group 1 according to an embodiment of the present invention
  • nucleus to be tested may be DNA and/or RNA, etc., and may also be referred to as “template strand” or “hybridization strand” in some embodiments, for example, in the process of detecting nucleic acid using a chip.
  • Target DNA strand may be DNA and/or RNA, etc., and may also be referred to as “template strand” or “hybridization strand” in some embodiments, for example, in the process of detecting nucleic acid using a chip.
  • the "probe” referred to in the embodiments of the present invention may be DNA and/or RNA, etc., and may also be referred to as "primer", “capture strand” or “fixed strand” in some embodiments.
  • the so-called “probes” can be randomly distributed or regularly distributed on the surface of the substrate, such as array distributions, such as capture chips currently on the market, where the probes are generally distributed in an array on the surface of the chip.
  • the so-called “substrate” can be any solid support that can be used to immobilize a nucleic acid sequence, such as a nylon membrane, a glass slide, a plastic, a silicon wafer, a magnetic bead, etc., and the surface of the chip can be used interchangeably with the surface of the substrate unless otherwise specified.
  • the probes are typically attached/immobilized on the surface of the substrate by chemical bonds.
  • the surface of the substrate is a chemically modified surface with reactive groups that can be attached to the probe. Surface modification, fixation, etc. can be carried out using known methods or directly customized or purchased.
  • non-specific adsorption generally refers to the adsorption caused by the force of non-covalent bonds, the force of non-covalent bonds Including hydrophobic interaction, van der Waals force, electrostatic force, etc.; on the chip with probe on the surface, in the process of nucleic acid detection based on the principle of base complementation, generally, the non-specific adsorption of nucleic acid refers to the non-covalent attachment of nucleic acid to the chip. On the surface and / or probe.
  • the so-called “marker” may be any physical, chemical or biologically detectable label.
  • the “signal” referred to in the embodiments of the present invention is a signal generated by the “marker” in a specific situation, typically but not limited.
  • a sexual "marker” is an optically detectable label, such as a fluorescent dye, and correspondingly, the “signal” is a fluorescent signal.
  • the fluorescent dyes are labeled, for example, Cy5 and/or Cy3, and both Cy5 and Cy3 are water-soluble 3H-phthalocyanine bioluminescent fluorescent dyes which can be emitted under laser irradiation at 650 nm and 550 nm, respectively. Red light color and green fluorescence.
  • test result can be any suitable qualitative or quantitative result, such as visual results, instrumental test results, and the like.
  • the non-specific adsorption of the nucleic acid to be tested can be judged by visually measuring the signal on the surface of the substrate, such as the distribution, intensity, etc. of the fluorescent signal.
  • the instrument/signal detection device can be used to detect the signal on the surface of the substrate to determine the non-specific adsorption of the nucleic acid to be tested.
  • the instrument can be an optical detection device with an imaging system, etc., including a light source. , objective lens and camera, correspondingly, the so-called “detection result” includes obtaining an image.
  • the so-called “bright spot” refers to the spot/spot on the image, and one spot/spot occupies at least one pixel.
  • the so-called “pixel” is the same as “pixel.”
  • the so-called “bright spot detection” corresponds to the detection of an optical signal of the nucleic acid or base or base cluster.
  • a method for detecting nucleic acid-specific and/or non-specific adsorption comprises: S100: reacting a nucleic acid to be tested with a first probe, the first probe being immobilized on a surface of the first substrate, to be tested The nucleic acid is at least partially complementary to the first probe, the nucleic acid to be tested carries a first label, the first probe carries a second label, the first label is capable of generating a first signal, and the second label is capable of generating a second signal; S200: detecting a signal on the surface of the first substrate to obtain a first detection result; S300: reacting the nucleic acid to be tested with the second probe, the second probe is fixed on the surface of the second substrate, and the second probe is provided with the second label, The nucleic acid is not complementary to the second probe; S400: detecting the signal on the surface of the second substrate to obtain a second detection result; S500: detecting the specificity of the nucleic acid to be tested
  • the non-specific and/or specific adsorption of the target nucleic acid by the probe on the surface of the substrate and/or the surface of the substrate can be qualitatively or quantitatively detected; the specificity can be qualitatively or quantitatively distinguished by detecting the signal on the surface of the substrate by the detection system.
  • non-specific adsorption obtaining information on the quantification and distribution of non-specific or specific adsorption, can be used in chip production and in all methods or applications involving chip detection of nucleic acid processes, such as for no-load chips (without probes) Or the evaluation of the performance of the capture chip (with probe), the quality control of the chip production, the prediction of the effect of the chip for capturing nucleic acid, the analysis and comparison, and the like.
  • the first signal and the second signal are detection-distinguishable signals, that is, the first signal and the second signal can be detected as two different signals, such as fluorescent signals of different colors.
  • S100 and S300 are performed in no order, and can be performed sequentially or simultaneously. Similarly, the same is true for S200 and S400. In some embodiments, S100 and S300 are parallel tests performed simultaneously.
  • the materials and surface characteristics of the first substrate and the second substrate in S100 and S300 are substantially the same or identical, such as hydrophilicity, surface size, and the like.
  • the first substrate surface and the second substrate surface are substrates of the same material properties that are subjected to the same surface treatment.
  • the first substrate surface and the second substrate surface are different surface regions of the same substrate that have been subjected to the same surface treatment.
  • the first substrate surface and the second substrate surface in S100 and S300 are different from the fixed probe, and other aspects such as surface size, probe immobilization density, probe distribution, amount of nucleic acid to be tested, reaction conditions, etc. Consistent.
  • the terms “substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the distribution or arrangement of the probes on the surface of the substrate is not limited.
  • the first probes are randomly distributed or regularly distributed on the surface of the first substrate (for example, distributed in an array), similarly, second.
  • the probes are randomly or regularly distributed on the surface of the second substrate (eg, in an array).
  • the distribution state of the second probe on the surface of the second substrate is the same as the distribution state of the first probe on the surface of the first substrate.
  • the first marker and the second marker can be different fluorescent dyes.
  • detecting a signal on a surface of the first substrate, obtaining the first detection result includes: taking a photo of the first substrate surface by the imaging system to obtain a first image; and detecting the first image to obtain the first Detecting the result; detecting the signal on the surface of the second substrate, obtaining the second detection result comprises: taking a picture of the second substrate surface by the imaging system to obtain a second image; and detecting the second image to obtain a second detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the first image to obtain the first detection result further comprises: detecting the first image to determine the number Na1 of positions at which the first signal and the second signal are simultaneously present on the surface of the first substrate and only There is a number Na3 of positions of the first signal; detecting the second image to obtain the second detection result further comprises: detecting the second image to determine the number Nb1 of positions at which the first signal and the second signal are simultaneously present on the surface of the second substrate And the number Nb3 of locations where only the first signal is present.
  • the image comprises a plurality of pixel points.
  • the description of the first image is taken as an example, and it should be understood that the following image detecting method is also applicable to the processing of the second image, and in the parallel test of the detection example of the same nucleic acid-specific/non-specific adsorption, Images of the test group and the control group, such as the same nucleic acid-specific/non-specific adsorption detection protocol, can be treated in the same manner to obtain reliable and comparable test results.
  • an image detecting method includes: an optional image pre-processing step S11, wherein the image pre-processing step S11 includes pre-processing the first image to obtain a first pre-processed image.
  • Image includes pre-processing the first image to obtain a first pre-processed image.
  • Bright spot detecting step S12 the bright spot detecting step S12 includes the steps of: S21, analyzing the first image to calculate a bright spot determination threshold, S22, analyzing the first image to obtain a candidate bright spot, and S23, determining whether the candidate bright spot is a bright spot according to the bright spot determination threshold.
  • the first image is processed by the image pre-processing step, and the calculation amount of the bright spot detecting step can be reduced.
  • whether the candidate bright spot is a bright spot is determined by the bright spot determining threshold, and the accuracy of determining the bright spot of the image can be improved.
  • the image pre-processing step S11 is a step taken to obtain a better detection effect.
  • the image may be directly subjected to a bright spot detection step.
  • the input first image to be detected may be a 16-bit tiff format image of 512*512 or 2048*2048, and the image of the tiff format may be a grayscale image. In this way, the processing of the image detecting method can be simplified.
  • the image pre-processing step S11 includes performing a background subtraction process on the first image to obtain a pre-processed first image. In this way, the noise of the first image can be further reduced, and the accuracy of the image detecting method is higher.
  • the image pre-processing step S11 includes: performing a simplification process on the first image subjected to the subtractive background processing to obtain a pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the image pre-processing step S11 includes filtering the first image to obtain a pre-processed first image. In this way, filtering the first image can obtain the pre-processed first image under the condition that the image detail features are retained as much as possible, thereby improving the accuracy of the image detection method.
  • the image pre-processing step S11 includes performing a subtractive background process on the first image and then performing a filtering process to obtain the pre-processed first image. In this way, after the background is subtracted from the first image and then filtered, the noise of the first image can be further reduced, and the accuracy of the image detection method is higher.
  • the image pre-processing step S11 includes: performing a simplification process on the first image after performing the subtractive background processing and then performing the filtering process to obtain the pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the image pre-processing step S11 includes performing a simplification process on the first image to obtain a pre-processed first image. In this way, the amount of calculation of the subsequent image detecting method can be reduced.
  • the step of determining whether the candidate bright spot is a bright spot according to the bright spot determination threshold includes: step S31, searching for the greater than (p*p-1) connectivity in the preprocessed first image. Pixel points and the found pixel points as the center of the candidate bright points, p*p and the bright points are in one-to-one correspondence, each value in p*p corresponds to one pixel point, p is a natural number and is an odd number greater than 1; S32.
  • I max can be understood as the center strongest intensity of the candidate bright spot.
  • I max is the strongest intensity of the center of the 3*3 window.
  • a BI is the ratio of the set value in the first image preprocessed in the 3*3 window, and ceof guass is the pixel and 2D of the 3*3 window. Correlation coefficient of Gaussian distribution.
  • the preprocessed first image is a simplified processed image.
  • the preprocessed first image may be a binarized image, that is, the set value in the binarized image may be that the pixel meets the set condition. The value corresponding to the time.
  • the binarized image may contain two values of 0 and 1 characterizing different attributes of the pixel, the set value is 1, and A BI is the ratio of 1 in the binarized image in the p*p window. .
  • the image includes a plurality of pixel points.
  • detecting the first image includes: highlighting the first image by using a k*k matrix.
  • the detecting comprises determining that the matrix whose central pixel value of the matrix is not less than any pixel value of the matrix non-center corresponds to a bright point, k is an odd number greater than 1, and the k*k matrix comprises k*k pixels.
  • the method is based on the difference between the brightness/intensity of the signal generated by the mark and the background brightness/intensity, enabling simple and rapid detection of the image, detection and acquisition of signal information.
  • the pixel value of the matrix center is greater than a first preset value, and any one of the matrix non-center pixel values is greater than the second pixel value.
  • the first preset value and the second preset value may be set according to experience or a certain amount of pixel/intensity data of a normal bright spot of a normal image, and the so-called "normal image” and "normal bright spot” may be normal to the naked eye. If the image looks clear, the background is clean, the size and intensity of the highlights are relatively uniform.
  • the first preset value and the second preset value are related to an average pixel value of the image. For example, the first preset value is set to be 1.4 times the average pixel value of the image, and the second preset value is 1.1 times the average pixel value of the image, which can eliminate interference and obtain a better bright spot detection result.
  • the first image is a color image
  • one pixel of the color image has three pixel values
  • the color image can be converted into a gray image, and then image detection is performed to reduce the calculation of the image detection process.
  • Quantity and complexity may be selected to be converted into a grayscale image using, but not limited to, a floating point algorithm, an integer method, a shift method, or an average value method.
  • the color image can also be directly detected.
  • the size comparison of the pixel values mentioned above can be regarded as a three-dimensional value or a comparison of the size of an array of three elements, and the relative sizes of the plurality of multi-dimensional values can be customized according to experience and needs, for example. When any two-dimensional value in the three-dimensional value a is larger than the value of the corresponding dimension of the three-dimensional value b, the three-dimensional value a is considered to be larger than the three-dimensional value b.
  • the first image is a grayscale image
  • the pixel value of the grayscale image is a one-dimensional value
  • the pixel value of the grayscale image is the same as the grayscale value
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified epoxy-bearing surface: a probe with a Cy3 fluorescent dye at the end.
  • Needle DNA capture strand-1, Capture DNA-1
  • the probe is an amino-modified probe with an amino group at the end, and the probe is reacted with the epoxy group on the surface of the chip by -NH 3 and immobilized in
  • the surface of the substrate, that is, the DNA capture strand-1 is attached to the surface of the chip by a chemical bond; then the surface of the chip is passivated using a passivation solution to block the unreacted epoxy group.
  • the template strand, the nucleic acid to be tested, is added, and the template strand is a DNA hybrid strand (Target DNA) having a Cy5 fluorescent dye molecule at its end and complementary to the DNA capture strand-1 base.
  • a fluorescence microscope such as a total internal reflection fluorescence microscope (TIRF) to obtain an image with signal points/spots.
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified epoxy group-bearing surface: the same reaction system and time are used to fix another probe.
  • the probe is a DNA strand (DNA capture strand-2, Capture DNA-2) with a Cy3 fluorescent dye label, the sequence of which is not complementary to the DNA hybrid strand.
  • Hybridization is carried out by adding the same hybrid template strand as described above, i.e., the nucleic acid to be tested. After the hybridization is completed, the surface is imaged by total internal reflection fluorescence microscopy (TIRF) to obtain an image with fluorescent signal spots/spots.
  • TIRF total internal reflection fluorescence microscopy
  • non-specific adsorption of the surface of the chip and/or the nucleic acid to be detected by the probe can be evaluated, for example, using the formula (Nb1+Nb3)/(Na1+Na3) Measuring the ratio of non-specific adsorption of nucleic acid on the surface of the first substrate and the first probe; determining the proportion of the non-specific adsorption of the nucleic acid to be detected in the first probe by using the formula Nb1/(Na1+Na3); using the formula (Na1+Na3-Nb1) -Nb3)/(Na1+Na3) determines the effective hybridization ratio of the test nucleic acid to the first probe.
  • a method for detecting non-specific adsorption of a nucleic acid comprises: S1000: reacting a nucleic acid to be detected with a third probe, and immobilizing a third probe on a surface of the third substrate, the nucleic acid to be tested a third mark, the third probe carries a fourth mark, the nucleic acid to be tested is not complementary to the third probe, the third mark is capable of generating a third signal, and the fourth mark is capable of generating a fourth signal; S2000: detecting the surface of the third substrate a signal on the third detection result; S3000: reacting the nucleic acid to be tested with the surface of the fourth substrate; S4000: detecting a signal on the surface of the fourth substrate to obtain a fourth detection result; S5000: based on the third detection result and the fourth The detection result detects the non-specific adsorption of the nucleic acid to be tested.
  • the non-specific adsorption of the target nucleic acid by the probe on the surface of the substrate and/or the surface of the substrate can be qualitatively or quantitatively detected; the non-specific adsorption can be qualitatively or quantitatively distinguished by detecting the signal on the surface of the substrate by the detection system, and non-specificity can be obtained.
  • Information on the amount of adsorption, distribution, etc. can be used in chip production and in all methods or applications involving chip detection of nucleic acid processes, such as evaluation of performance of no-load chips (without probes) or capture chips (with probes) Quality control of chip production, prediction, analysis and comparison of effects of chip capture nucleic acid.
  • the third signal and the fourth signal are detection-distinguishable signals, that is, the third signal and the fourth signal can be detected as two different signals, such as fluorescent signals of different colors.
  • the S1000 and S3000 are carried out in no order, and can be carried out sequentially or simultaneously. Similarly, the S2000 and S4000 are also carried out without order. In some embodiments, S1000 and S3000 are parallel tests, performed simultaneously.
  • the materials and surface characteristics of the third and fourth substrates in S1000 and S3000 are substantially the same or identical, such as hydrophilicity, surface size, and the like.
  • the third substrate surface and the fourth substrate surface are substrates of the same surface properties that are subjected to the same surface treatment.
  • the third substrate surface and the fourth substrate surface are different surface regions of the same substrate that have been subjected to the same surface treatment.
  • the third substrate surface and the fourth substrate surface in S1000 and S3000 are substantially identical except for whether or not the probe is immobilized, such as surface size, amount of nucleic acid to be tested, reaction conditions, and the like.
  • the fourth substrate surface is distinguished from the third substrate surface by a fixed probe thereon, for example, it may be an empty substrate surface, i.e., a substrate surface without a probe.
  • a fixed probe thereon, for example, it may be an empty substrate surface, i.e., a substrate surface without a probe.
  • the distribution or arrangement of the third probes on the surface of the third substrate is not limited. In the embodiment of the invention, the third probes are randomly distributed or regularly distributed on the surface of the third substrate (for example, in an array).
  • the first marker and the second marker are different fluorescent dyes.
  • detecting a signal on a surface of the third substrate, obtaining a third detection result includes: photographing a third substrate surface by an imaging system to obtain a third image; and detecting the third image to obtain a third Detecting the result; detecting the signal on the surface of the fourth substrate, obtaining the fourth detection result comprises: taking a photo of the fourth substrate surface by the imaging system to obtain a fourth image; and detecting the fourth image to obtain a fourth detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the third image to obtain the third detection result further comprises: detecting the third image to determine the number Nc3 of positions where only the third signal exists on the surface of the third substrate; detecting the fourth image, Obtaining the fourth detection result further includes detecting the fourth image to determine the number Nd of locations where the third signal is present on the surface of the fourth substrate.
  • the detection of the image can be referred to the above-described detection example of the first image.
  • the non-specific adsorption of the nucleic acid to be detected before and after the immobilization of the probe on the surface of the chip can be determined by using the formula (Nc3-Nd)/Nc3 to realize non-specific adsorption of the nucleic acid to be detected based on the third detection result and the fourth detection result.
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified epoxy-bearing surface: a probe with a Cy3 fluorescent dye at the end.
  • the needle DNA capture strand-2, Capture DNA-2
  • the probe is a terminal amino-modified DNA single strand, which is modified by -NH 3 and the surface of the chip.
  • the group reacts to attach the DNA capture strand-2 to the surface of the chip by a chemical bond; then the passivation layer is used to passivate the surface of the chip to block the unreacted epoxy group.
  • a hybrid template strand that is, a nucleic acid to be tested, and a template chain having a Cy5 fluorescent dye molecule at the end is added to carry out hybridization.
  • the surface is photographed by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • the surface of the substrate is also referred to as a chip surface, and is a surface modified with an epoxy group, which is the same as the surface of the substrate except that the probe is not included, the reaction system and The reaction time is the same as the example of Fig. 4 described above.
  • the hybrid template strand, the nucleic acid to be tested is further added for hybridization.
  • the surface can be imaged by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • a method for detecting nucleic acid-specific and/or non-specific adsorption comprises: S10000: reacting a nucleic acid to be tested with a fifth probe, and fixing a fifth probe on a surface of the fifth substrate, The nucleic acid is at least partially complementary to the fifth probe, the nucleic acid to be tested carries a fifth label, the fifth probe carries a sixth label, the fifth label is capable of generating a fifth signal, and the sixth label is capable of generating a sixth signal; S20000: Detecting a signal on the surface of the fifth substrate to obtain a fifth detection result; S30000: reacting the nucleic acid to be tested with the sixth probe, the sixth probe is fixed on the surface of the sixth substrate, and the sixth probe is marked with a sixth mark, The nucleic acid to be tested is not complementary to the sixth probe; S40000: detecting the signal on the surface of the sixth substrate to obtain a sixth detection result; S50000: reacting the nucleic acid
  • the non-specific and/or specific adsorption of the target nucleic acid by the probe on the surface of the substrate and/or the surface of the substrate can be qualitatively or quantitatively detected; the specificity can be qualitatively or quantitatively distinguished by detecting the signal on the surface of the substrate by the detection system.
  • non-specific adsorption obtaining information on the quantification and distribution of non-specific or specific adsorption, can be used in chip production and in all methods or applications involving chip detection of nucleic acid processes, such as for no-load chips (without probes) Or the evaluation of the performance of the capture chip (with probe), the quality control of the chip production, the prediction of the effect of the chip for capturing nucleic acid, the analysis and comparison, and the like.
  • the fifth signal and the sixth signal are detection-distinguishable signals, that is, the fifth signal and the sixth signal can be detected as two different signals, such as fluorescent signals of different colors.
  • S10000, S30000, and S50000 are performed in no order, and may be performed sequentially or simultaneously. Similarly, the execution of S20000, S40000, and S60000 is also not limited in order. In some embodiments, S10000, S30000, and S50000 are parallel tests, performed simultaneously.
  • the materials, surface characteristics such as hydrophilicity, surface size, and the like of the fifth, sixth, and seventh substrates in S10000, S30000, and S50000 are substantially the same or identical.
  • the fifth substrate, the sixth substrate, and the seventh substrate are substrates of the same surface properties that are subjected to the same surface treatment.
  • the fifth substrate, the sixth substrate, and the seventh substrate are different surface regions of the same substrate that have been subjected to the same surface treatment.
  • the fifth substrate surface and the sixth substrate surface in S10000 and S30000 are different from the fixed probe, and other aspects such as surface size, probe immobilization density, probe distribution, amount of nucleic acid to be tested, reaction conditions, etc. Consistent.
  • the seventh substrate in S50000 has substantially the same as the fifth substrate surface and the sixth substrate in S10000 and S30000, except for the absence of the immobilization probe, such as the surface size, the amount of the nucleic acid to be tested, the reaction conditions, and the like.
  • the terms “substantially identical”, “substantially identical” and “consistent” or “identical” mean that the differences produced by different batch preparation, processing, and/or parallel tests are within the allowable tolerances.
  • the distribution or arrangement of the probes on the surface of the substrate is not limited.
  • the fifth probes are randomly distributed or regularly distributed on the surface of the fifth substrate (for example, in an array), similarly, the sixth The probes are randomly distributed or regularly distributed (eg, in an array) on the surface of the sixth substrate.
  • the distribution state of the sixth probe on the surface of the sixth substrate was the same as that of the fifth probe on the surface of the fifth substrate.
  • the fifth and sixth markers can be different fluorescent dyes.
  • detecting a signal on a surface of the fifth substrate, obtaining a fifth detection result includes: photographing a fifth substrate surface by an imaging system to obtain a fifth image; and detecting the fifth image to obtain a fifth Test results.
  • Detecting a signal on the surface of the sixth substrate, obtaining the sixth detection result includes: taking a picture of the sixth substrate surface by the imaging system to obtain a sixth image; and detecting the sixth image to obtain a sixth detection result.
  • Detecting a signal on the surface of the seventh substrate, obtaining the seventh detection result includes: photographing the surface of the seventh substrate by the imaging system to obtain a seventh image; and detecting the seventh image to obtain a seventh detection result.
  • the imaging system may be an optical detection device or the like with an imaging system, including a light source, an objective lens, and a camera.
  • detecting the fifth image to obtain the fifth detection result further comprises: detecting the fifth image to determine the number Ne1 of the positions of the fifth signal and the sixth signal simultaneously on the surface of the fifth substrate and only There is a number Ne3 of positions of the fifth signal.
  • Detecting the sixth image to obtain the sixth detection result further includes: detecting the sixth image to determine the number Nf1 of positions at which the fifth signal and the sixth signal exist simultaneously on the sixth substrate surface, and the number of positions where only the fifth signal exists Nf3.
  • Detecting the seventh image to obtain the seventh detection result further includes detecting the seventh image to determine the number Ng of locations where the fifth signal is present on the surface of the seventh substrate.
  • the detection of the image can be referred to the above-described detection example of the first image.
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified epoxy-bearing surface: a probe with a Cy3 fluorescent dye at the end.
  • Needle DNA capture strand-1, Capture DNA-1
  • the probe is an amino-modified probe with an amino group at the end, and the probe is reacted with the epoxy group on the surface of the chip by -NH 3 and immobilized in
  • the surface of the substrate, that is, the DNA capture strand-1 is attached to the surface of the chip by a chemical bond; then the surface of the chip is passivated using a passivation solution to block the unreacted epoxy group.
  • the template strand, the nucleic acid to be tested, is added, and the template strand is a DNA hybrid strand (Target DNA) having a Cy5 fluorescent dye molecule at its end and complementary to the DNA capture strand-1 base.
  • a fluorescence microscope such as a total internal reflection fluorescence microscope (TIRF) to obtain an image with signal points/spots.
  • the surface of the substrate also referred to as the surface of the chip, is a surface-modified epoxy group-bearing surface: the same reaction system and time are used to fix another probe.
  • the probe is a DNA strand (DNA capture strand-2, Capture DNA-2) with a Cy3 fluorescent dye label, the sequence of which is not complementary to the DNA hybrid strand.
  • Hybridization is carried out by adding the same hybrid template strand as described above, i.e., the nucleic acid to be tested. After the hybridization is completed, the surface is imaged by total internal reflection fluorescence microscopy (TIRF) to obtain an image with fluorescent signal spots/spots.
  • TIRF total internal reflection fluorescence microscopy
  • the surface of the substrate is also referred to as a chip surface, and is a surface modified with an epoxy group, which is the same as the surface of the substrate except that the probe is not included, the reaction system and The reaction time is the same as the example of Fig. 4 described above.
  • the hybrid template strand, the nucleic acid to be tested is further added for hybridization.
  • the surface can be imaged by a fluorescence microscope including a total internal reflection fluorescence microscope (TIRF) to obtain an image with a fluorescent signal.
  • TIRF total internal reflection fluorescence microscope
  • Example 1 Investigating the effects of non-specific adsorption during DNA hybridization
  • Chip glass with epoxy group on the surface (purchased from SCHOTT);
  • EKB-6P-Cy3 Fixed strand 1 (EKB-6P-Cy3): a specific sequence of artificial synthesis, with a short DNA sequence of -NH 3 and a fluorescent group Cy3 dye at the end, the sequence is specifically:
  • EKB-7P-Cy3 Fixed strand 2
  • a synthetically specific sequence with a short DNA sequence of -NH 3 and a fluorophore Cy3 dye at the end, the sequence being:
  • Hybrid strand (EKB-6T-Cy5): a synthetically complementary pair with a fixed strand 1 and a non-complementary pair with a fixed strand 2, with a sequence of 35 bp in length and a short DNA sequence with a fluorescent group Cy5 at the end, sequence Specifically:
  • Control group 1 After washing and drying the glass, the reaction was carried out at 37 ° C in a solution containing 150 mM K 2 HPO 4 and containing an amino-modified fixed chain 2 (EKB-7P-Cy3) nucleic acid probe at a concentration of 1.0 M. After 0.5 hours, it was washed successively with 3X SSC solution (containing 0.1% Triton), 3XSSC, 0.15 MK 2 HPO 4 solution, and then passivated with 1 M K 2 HPO 4 at 37 ° C for 17 hours.
  • 3X SSC solution containing 0.1% Triton
  • 3XSSC 0.15 MK 2 HPO 4 solution
  • Control group 2 After the glass was washed and dried, it was placed in a 150 mM K 2 HPO 4 solution, and reacted at 37 ° C for 0.5 hour, and sequentially washed with 3X SSC solution (containing 0.1% Triton), 3X SSC, 0.15 M K 2 HPO 4 solution. Thereafter, 1 M K 2 HPO 4 was added and passivated at 37 ° C for 17 hours.
  • 3X SSC solution containing 0.1% Triton
  • 3X SSC 0.15 M K 2 HPO 4 solution.
  • 1 M K 2 HPO 4 was added and passivated at 37 ° C for 17 hours.
  • Chip (Flow Cell) Assembly Assemble the passivated glass into other multi-channel chips with other substrates or substrates.
  • Hybridization of hybridization The assembled multi-channel flow cell was added to Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS), reconstituted at 55 ° C for 0.5 hour, and then the concentration of hybridization chain (EKB-6T-Cy5) was added. A 1 nM solution of 3XSSC was reacted at 55 ° C for 0.5 hours, and then the channels were washed successively using Rinse buffer (1XSSC + 150 mM HEPES + 0.1% SDS) and Buffer H (150 mM HEPES + 150 mM NaCl).
  • Photographing Photographing the surface of the chip while using TIRF.
  • the image is detected using the method of the above example, including identifying, locating, and counting bright spots/bright spots, and the results are as follows:
  • the results of the experimental group are shown in Fig. 6.
  • the left image shows the Cy3 fluorescence point in one field of view, and the right image shows the Cy5 fluorescence point in the same field of view with a coincidence ratio of about 80%.
  • Fig. 7 The results of the control group 1 are shown in Fig. 7, in which the left panel shows the Cy3 fluorescence dots in one field of view, and the right panel shows the Cy5 fluorescence dots in the same field of view, in which the coincidence ratio is.

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Abstract

一种检测核酸特异性和/或非特异性吸附的方法，包括：使待测核酸与第一探针反应，第一探针固定在第一基底表面上，待测核酸与第一探针至少部分互补，待测核酸和第一探针带有不同的标记，标记能够产生信号；检测第一基底表面上的信号，获得第一检测结果；使待测核酸与第二探针反应，第二探针固定在第二基底表面上，第二探针带有第二标记，待测核酸与第二探针不互补；检测第二基底表面上的信号，获得第二检测结果；基于第一检测结果和第二检测结果，检测待测核酸的特异性和/或非特异性吸附。该方法能够对核酸与基底表面或基底表面上的探针的特异性和/或非特异性吸附情况进行检测，可用于芯片的生产质控中。

Description

检测核酸特异性和/或非特异性吸附的方法 技术领域
本发明涉及核酸检测技术领域，具体涉及一种检测核酸特异性和/或非特异性吸附的方法。
背景技术
在芯片上进行核酸检测，例如利用表面固定有探针的芯片捕获核酸以及进一步对捕获得的核酸进行分析检测，包括序列测定、信息分析等，芯片表面和/或其上的探针对核酸的非特异性吸附，会影响获得的有效数据量以及检测结果的准确性。对非特异性吸附情况的定性或定量检测，能够判定芯片的质量、芯片表面修饰情况以及用于检测结果预测等。
例如在目前的核酸测序中，特别是利用芯片的基因测序中，在芯片上进行核酸杂交时存在非特异性情况，包括芯片表面对模板链的非特异性吸附和芯片表面上的探针对模板链的非特异性吸附。这些非特异性吸附会造成测序结果错误率增加，导致测序通量和测序质量的下降。
因此，评估这些非特异性吸附情况，对于芯片生产质控、利用芯片进行核酸检测等领域具有重要意义。
发明内容
本发明实施例旨在至少解决现有技术中存在的技术问题之一或者至少提供一种有用的商业选择。为此，本发明提供一种检测核酸特异性和/或非特异性吸附的方法。
根据第一方面，提供一种检测核酸特异性和/或非特异性吸附的方法，该方法包括：使待测核酸与第一探针反应，上述第一探针固定在第一基底表面上，上述待测核酸与上述第一探针至少部分互补，上述待测核酸带有第一标记，上述第一探针带有第二标记，上述第一标记能够产生第一信号，上述第二标记能够产生第二信号；检测上述第一基底表面上的信号，获得第一检测结果；使上述待测核酸与第二探针反应，上述第二探针固定在第二基底表面上，上述第二探针带有上述第二标记，上述待测核酸与上述第二探针不互补；检测上述第二基底表面上的信号，获得第二检测结果；基于上述第一检测结果和上述第二检测结果，检测上述待测核酸的特异性和/或非特异性吸附。
根据第二方面，提供一种检测核酸非特异性吸附的方法，该方法包括：使待测核酸与第三探针反应，上述第三探针固定在第三基底表面上，上述待测核酸带有第三标记，上述第三探针带有第四标记，上述待测核酸与上述第三探针不互补，上述第三标记能够 产生第三信号，上述第四标记能够产生第四信号；检测上述第三基底表面上的信号，获得第三检测结果；使待测核酸与第四基底表面反应；检测上述第四基底表面上的信号，获得第四检测结果；基于上述第三检测结果和上述第四检测结果，检测上述待测核酸的非特异性吸附。
根据第三方面，提供一种检测核酸特异性和/或非特异性吸附的方法，该方法包括：使待测核酸与第五探针反应，第五探针固定在第五基底表面上，待测核酸与第五探针至少部分互补，待测核酸带有第五标记，上述第五探针带有第六标记，上述第五标记能够产生第五信号，上述第六标记能够产生第六信号；检测上述第五基底表面上的信号，获得第五检测结果；使上述待测核酸与第六探针反应，上述第六探针固定在第六基底表面上，上述第六探针带有上述第六标记，上述待测核酸与上述第六探针不互补；检测上述第六基底表面上的信号，获得第六检测结果；使待测核酸与第七基底表面反应；检测上述第七基底表面上的信号，获得第七检测结果；基于上述第五检测结果、第六检测结果和第七检测结果中的至少两个检测结果，检测上述待测核酸的特异性和/或非特异性吸附。
上述检测核酸非特异性吸附的方法，能够定性或定量检测基底表面和/或基底表面上的探针对目标核酸的非特异吸附情况和/或特异性吸附情况；通过检测系统检测基底表面的信号，能够定性或定量区分特异性和非特异性吸附，获得非特异性或特异性吸附的定量、分布情况等信息，能够用于芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片捕获核酸的效果的预测、分析比较等。
附图说明
图1为本发明实施方式中的图像处理方法的流程示意图；
图2为本发明实施方式中的图像处理方法的另一流程示意图；
图3为本发明实施方式中一个基底表面上探针固定和待测核酸杂交原理示意图；
图4为本发明实施方式中另一个基底表面上探针固定和待测核酸杂交原理示意图；
图5为本发明实施方式中又一个基底表面上待测核酸杂交原理示意图；
图6为本发明实施例中实验组拍照获得的一个视野中的Cy3荧光点(左图)和Cy5荧光点(右图)图像；
图7为本发明实施例中对照组1拍照获得的一个视野中的Cy3荧光点(左图)和Cy5荧光点(右图)图像；
图8为本发明实施例中对照组2拍照获得的一个视野中的Cy3荧光点(左图)和Cy5荧光点(右图)图像。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员不需创造性劳动可以认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。本领域技术人员能够根据说明书中的描述以及本领域的一般技术知识实现相关操作。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
为便于理解，以下结合附图通过举例的方式对本发明实施例进行详细说明。应当理解的是，如下举例中的对具体操作及操作细节的描述为示意，非对发明方案的限定。
在本发明的描述中，需要理解的是，术语“第一”、“第二”、“第三”、“第四”等仅用于描述目的，而不能理解为指示或暗示相对重要性、相对顺序或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”、“第三”、“第四”等的特征可以明示或者隐含地包括一个或者更多个所述特征。在本发明的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。本发明实施方式所称的“待测核酸”可以是DNA和/或RNA等，在一些实施方式中也可称为“模板链”或“杂交链”，例如，在利用芯片检测核酸过程中的目标DNA链。
本发明实施方式所称的“探针”可以是DNA和/或RNA等，在一些实施方式中也可称为“引物”、“捕获链”或“固定链”。所称的“探针”可以在基底表面上随机分布或者规则分布，如阵列分布，例如在目前市面上的捕获芯片，探针在芯片表面上一般呈阵列分布。
所称的“基底”可以是任何可用于固定核酸序列的固体支持物，例如尼龙膜、玻璃片、塑料、硅片、磁珠等，如无特殊说明，芯片表面与基底表面可互换使用。固相探针，探针一般是通过化学键连接/固定在基底表面上，一般地，基底表面为经过化学修饰的表面，带有反应基团，可与探针连接。表面修饰、固定等可利用已知方法进行或者直接定制或者购买。
所称的“非特异性吸附”和“特异性吸附”/“特异性结合”是相对的，“非特异性吸附”一般是指非共价键的作用力导致的吸附，非共价键的作用力包括疏水作用力、范德华力、静电作用力等；在表面带有探针的芯片上、基于碱基互补原则的核酸检测过程中，一般地，核酸非特异性吸附指核酸非共价的连接在芯片表面和/或探针上。
所称的“标记”可以是任何物理、化学或生物可检测标记，相应的，本发明实施方式所称的“信号”，是所述“标记”在特定情形下产生的信号，典型但非限定性的“标记”是光学可检测标记，例如荧光染料，相应的，所述“信号”是荧光信号。在本发明一些实施例中，标记为荧光染料，例如为Cy5和/或Cy3，Cy5和Cy3都属于水溶性3H-吲哚菁型生物荧光标示染料，分别能够在650nm和550nm的激光照射下发出红光色荧光和绿色荧光。
所称的“检测结果”可以是任何合适的定性或定量结果，例如目测结果、仪器检测结果等。在一些不需要精确定量的应用场景下，可以通过目测基底表面上的信号，例如荧光信号的分布、强度等来判断待测核酸的非特异性吸附情况。在一些需要精确定量的应用场景下，可以借助仪器/信号检测装置检测基底表面上的信号来判断待测核酸的非特异性吸附情况，例如仪器可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机，相应的，所称的“检测结果”包括获得图像。
所称的“亮点”，指图像上的光点/光斑，一个光点/光斑占有至少一个像素点。所称“像素点”同“像素”。利用光学检测系统对带有荧光标记的核酸进行检测时，所称的“亮点检测”对应于对该核酸或者碱基或碱基簇的光学信号的检测。
在本发明的一个实施例中，检测核酸特异性和/或非特异性吸附的方法包括：S100：使待测核酸与第一探针反应，第一探针固定在第一基底表面上，待测核酸与第一探针至少部分互补，待测核酸带有第一标记，第一探针带有第二标记，第一标记能够产生第一信号，第二标记能够产生第二信号；S200：检测第一基底表面上的信号，获得第一检测结果；S300：使待测核酸与第二探针反应，第二探针固定在第二基底表面上，第二探针带有第二标记，待测核酸与第二探针不互补；S400：检测第二基底表面上的信号，获得第二检测结果；S500：基于第一检测结果和第二检测结果，检测待测核酸的特异性和/或非特异性吸附。
利用该方法，能够定性或定量检测基底表面和/或基底表面上的探针对目标核酸的非特异和/或特异性吸附情况；通过检测系统检测基底表面的信号，能够定性或定量区分特异性和非特异性吸附，获得非特异性或特异性吸附的定量、分布情况等信息，能够用 于芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片捕获核酸的效果的预测、分析比较等。
上述方法中，第一信号和第二信号是检测上可区分的信号，即能够检测到第一信号和第二信号是两个不同的信号，例如不同颜色的荧光信号等。
S100和S300的进行无顺序要求，可先后进行，也可同时进行。类似地，S200和S400也是如此。在一些实施例中，S100和S300为平行试验，同时进行。
在一平行试验示例中，S100和S300中的第一基底和第二基底的材质、表面特性如亲疏水、表面大小等是基本相同或一致的。在一个例子中，第一基底表面和第二基底表面是经过相同表面处理的、相同材料性能的基底。在另一个例子中，第一基底表面和第二基底表面是经过相同表面处理的同一基底的不同表面区域。例如，S100和S300中的第一基底表面和第二基底表面除了所固定的探针不一样，其它方面如表面大小、探针固定密度、探针分布、待测核酸的量、反应条件等基本一致。所称的“基本一致”、“基本相同”同“一致”或“相同”，指不同批次制备、处理和/或平行试验产生的差异在允许的偏差范围内。
对探针在基底表面上的分布或排布方式不作限制，在本发明实施例中，第一探针在第一基底表面上随机分布或规则分布(例如呈阵列分布)，类似地，第二探针在第二基底表面上随机分布或规则分布(例如呈阵列分布)。在平行实验中，第二探针在第二基底表面上的分布状态与第一探针在第一基底表面上的分布状态相同。
第一标记和第二标记可以为不同的荧光染料。在本发明的实施例中，检测第一基底表面上的信号，获得第一检测结果包括：通过成像系统对第一基底表面进行拍照，获得第一图像；以及检测第一图像，以获得第一检测结果；检测第二基底表面上的信号，获得第二检测结果包括：通过成像系统对第二基底表面进行拍照，获得第二图像；以及检测第二图像，以获得第二检测结果。成像系统可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机。
在本发明的实施例中，检测第一图像，以获得第一检测结果进一步包括：检测第一图像，以确定第一基底表面上同时存在第一信号和第二信号的位置的数量Na1以及只存在第一信号的位置的数量Na3；检测第二图像，以获得第二检测结果进一步包括：检测第二图像，以确定第二基底表面上同时存在第一信号和第二信号的位置的数量Nb1以及只存在第一信号的位置的数量Nb3。
进一步，进行以下(a)，(b)和(c)中的至少之一，来实现基于第一检测结果和第二检测结果，检测待测核酸的特异性和/或非特异性吸附：(a)利用公式(Nb1+Nb3)/(Na1+Na3)确定待测核酸非特异性吸附在第一基底表面和第一探针上的比例；(b)利用公式Nb1/(Na1+Na3)确定待测核酸非特异性吸附在第一探针上的比例；(c)利用公式(Na1+Na3-Nb1-Nb3)/(Na1+Na3)确定待测核酸与第一探针特异性结合的比例。
以下结合附图说明示例图像的检测方法。本发明实施例中，图像包含多个像素点。以下示例描述中，以第一图像的检测为例进行描述，应当理解以下图像检测方法同样也适用于第二图像的处理，并且在同一核酸特异性/非特异性吸附的检测示例的平行试验中，如同一核酸特异性/非特异性吸附的检测方案的试验组和对照组的图像，均可采用相同的处理方式，以获得可信的、可对比的检测结果。
如图1所示，在本发明的一个的实施例中，图像检测方法，包括：可选的图像预处理步骤S11，图像预处理步骤S11包括预处理第一图像以获得预处理后的第一图像；亮点检测步骤S12，亮点检测步骤S12包括步骤：S21，分析第一图像以计算亮点判定阈值，S22，分析第一图像以获取候选亮点，S23，根据亮点判定阈值判断候选亮点是否为亮点。
上述图像检测方法，通过图像预处理步骤对第一图像进行处理，可减少亮点检测步骤的计算量，同时，通过亮点判断阈值判断候选亮点是否为亮点，可提高判断图像亮点的准确性。需要说明的是，图像预处理步骤S11是为了获得更好的检测效果所采用的步骤，在其它实施方式中，可以直接对图像进行亮点检测步骤。
具体地，在一个例子中，输入的待检测的第一图像可为512*512或2048*2048的16位tiff格式的图像，tiff格式的图像可为灰度图像。如此，可简化图像检测方法的处理过程。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行减背景处理，以获得预处理的第一图像。如此，能够进一步减少第一图像的噪声，使图像检测方法的准确性更高。
在某些实施方式中，图像预处理步骤S11包括：对进行减背景处理后的第一图像进行简化处理，以获得预处理的第一图像。如此，可减少后续图像检测方法的计算量。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行滤波处理，以获得预处理后的第一图像。如此，对第一图像进行滤波可在尽量保留图像细节特征的条件下 获取预处理的第一图像，进而可提高图像检测方法的准确性。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行减背景处理后再进行滤波处理，以获得预处理后的第一图像。如此，对第一图像进行减背景后再进行滤波，能够进一步减少第一图像的噪声，使图像检测方法的准确性更高。
在某些实施方式中，图像预处理步骤S11包括：对进行减背景处理后再进行滤波处理后的第一图像进行简化处理，以获得预处理的第一图像。如此，可减少后续图像检测方法的计算量。
在某些实施方式中，图像预处理步骤S11包括：对第一图像进行简化处理以获得预处理的第一图像。如此，可减少后续图像检测方法的计算量。
在某些实施方式中，请参图2，根据亮点判定阈值判断候选亮点是否为亮点的步骤，包括：步骤S31，在预处理后的第一图像中查找大于(p*p-1)连通的像素点并将查找到的像素点作为候选亮点的中心，p*p与亮点是一一对应的，p*p中的每个值对应一个像素点，p为自然数且为大于1的奇数；步骤S32，判断候选亮点的中心是否满足条件：I max*A BI*ceof guass＞T，其中，I max为p*p窗口的中心最强强度，A BI为p*p窗口中预处理后的第一图像中为设定值所占的比率，ceof guass为p*p窗口的像素和二维高斯分布的相关系数，T为亮点判定阈值。若满足上述条件，S33，判断候选亮点的中心对应的亮点为待处理图像所包含的亮点；若不满足上述条件，S34，弃去候选亮点的中心对应的亮点。如此，实现了亮点的检测。
具体地，I max可理解为候选亮点的中心最强强度。在一个例子中，p＝3，查找大于8连通的像素点。将查找到的像素点作为候选亮点的像素点。I max为3*3窗口的中心最强强度，A BI为3*3窗口中预处理后的第一图像中为设定值所占的比率，ceof guass为3*3窗口的像素和二维高斯分布的相关系数。
预处理后的第一图像为简化处理后的图像，例如预处理后的第一图像可为二值化图像，也就是说，二值化图像中的设定值可为像素点满足设定条件时所对应的值。在另一个例子中，二值化图像可包含表征像素点不同属性的0和1二个数值，设定值为1，A BI为p*p窗口中二值化图像中为1所占的比率。
在本发明的另一个实施例中，对于拍照得的图像的检测，图像均包含多个像素点，以第一图像为例，检测第一图像包括：利用k*k矩阵对第一图像进行亮点检测，包括判 定该矩阵的中心像素值不小于该矩阵非中心任一像素值的矩阵对应一个亮点，k为大于1的奇数，k*k矩阵包含k*k个像素点。该方法基于标记所产生的信号的亮度/强度与背景亮度/强度的差异，能够简单快速的检测图像，检测到以及获得信号的信息。
在某些实施例中，所述矩阵中心的像素值大于第一预设值，所述矩阵非中心任一像素值大于第二像素值。
第一预设值和第二预设值可以根据经验或者一定量的正常图像的正常亮点的像素/强度数据来设定，所称的“正常图像”、“正常亮点”可以是肉眼看起来正常，如图像看起来清晰、背景较干净，亮点大小及强度较均匀等。在一个实施例中，第一预设值和第二预设值与该图像的平均像素值相关。例如，设定第一预设值为该图像的平均像素值的1.4倍，第二预设值为该图像的平均像素值的1.1倍，能够排除干扰、获得较佳的亮点检测结果。
具体地，在一个示例中，第一图像是彩色图像，彩色图像的一个像素点具有三个像素值，可以将该彩色图像转化为灰度图像，再进行图像检测，以降低图像检测过程的计算量和复杂度。可选择但不限于利用浮点算法、整数方法、移位方法或平均值法等将非灰度图像转换成灰度图像。当然，也可以直接检测彩色图像，上述涉及的像素值的大小比较可看成是三维值或者具有三个元素的数组的大小比较，可根据经验及需要自定义多个多维值的相对大小，例如当三维值a中的任两维数值比三维值b的对应维度的数值大，可认为三维值a大于三维值b。
在另一个示例中，第一图像是灰度图像，灰度图像的像素值为一维数值，灰度图像的像素值同灰度值。
在一个例子中，如图3所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：加入末端带有Cy3荧光染料标记的探针(DNA捕获链-1，Capture DNA-1)，探针为经过氨基化修饰的探针、末端带有氨基基团，探针通过-NH 3与芯片表面的环氧基团反应，固定在基底表面，即将DNA捕获链-1通过化学键连接到芯片表面；然后使用钝化液钝化芯片表面，以封闭未反应的环氧基团。
加入模板链，即待测核酸，模板链为末端带有Cy5荧光染料分子并与DNA捕获链-1碱基互补配对的DNA杂交链(Target DNA)。杂交完成后，通过荧光显微镜例如全内反射荧光显微镜(TIRF)对表面进行拍照，可得到带有信号点/斑的图像。(1)如果同一个位置观察到两种荧光信号，则说明该位置有固定杂交的DNA双链或者存在部分少量的链上吸附，数量Na1；(2)如果只观察到绿色荧光信号，则说明该位置只有捕 获链而没有杂交链，数量Na2；(3)如果只观察到红色荧光信号，则说明该位置只有杂交链，则为杂交链在基底表面的非特异性吸附以及极少量捕获链荧光猝灭的情况，数量Na3。
如图4所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：利用相同的反应体系及时间，将另一种探针固定在该表面上，该探针为带有Cy3荧光染料标记的DNA链(DNA捕获链-2，Capture DNA-2)，其序列与DNA杂交链不互补。
加入与上述相同的杂交模板链，即待测核酸，进行杂交。杂交完成后，通过全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号点/斑的图像。(1)如果在同一个位置观察到两种荧光信号，则说明该位置有DNA双链同时存在，则为杂交模板链的链上特异性吸附，数量Nb1；(2)如果只观察到绿色荧光信号，则说明该位置只有捕获链而没有杂交链，数量Nb2；(3)如果只观察到红色荧光信号，则说明该位置只有杂交链，为杂交链在基底表面的非特异性吸附，数量Nb3。
基于上述信息，可进行量化区分特异性吸附和非特异性吸附，评估芯片表面和/或探针对待测核酸的非特异性吸附等，例如利用公式(Nb1+Nb3)/(Na1+Na3)可以确定待测核酸非特异性吸附在第一基底表面和第一探针的比例；利用公式Nb1/(Na1+Na3)确定待测核酸非特异性吸附在第一探针的比例；利用公式(Na1+Na3-Nb1-Nb3)/(Na1+Na3)确定待测核酸与第一探针的有效杂交比例。
在本发明的另一个实施例中，检测核酸非特异性吸附的方法，包括：S1000：使待测核酸与第三探针反应，第三探针固定在第三基底表面上，待测核酸带有第三标记，第三探针带有第四标记，待测核酸与第三探针不互补，第三标记能够产生第三信号，第四标记能够产生第四信号；S2000：检测第三基底表面上的信号，获得第三检测结果；S3000：使待测核酸与第四基底表面反应；S4000：检测第四基底表面上的信号，获得第四检测结果；S5000：基于第三检测结果和第四检测结果，检测待测核酸的非特异性吸附。
利用该方法，能够定性或定量检测基底表面和/或基底表面上的探针对目标核酸的非特异吸附情况；通过检测系统检测基底表面的信号，能够定性或定量区分非特异性吸附，获得非特异性吸附的定量、分布情况等信息，能够用于芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片捕获核酸的效果的预测、分析比较等。
上述方法中，第三信号和第四信号是检测上可区分的信号，即能够检测到第三信号 和第四信号是两个不同的信号，例如不同颜色的荧光信号等。
S1000和S3000的进行无顺序要求，可先后进行，也可同时进行。同样的，S2000和S4000的进行也无顺序限制。在一些实施例中，S1000和S3000是平行试验，为同时进行。
在一平行试验示例中，S1000和S3000中的第三基底和第四基底的材质、表面特性如亲疏水、表面大小等是基本相同或一致的。在一个例子中，第三基底表面和第四基底表面是经过相同表面处理的、相同材料性能的基底。在另一个例子中，第三基底表面和第四基底表面是经过相同表面处理的同一基底的不同表面区域。例如，S1000和S3000中的第三基底表面和第四基底表面除了是否固定探针以外，其它方面如表面大小、待测核酸的量、反应条件等基本一致。第四基底表面区别于第三基底表面的是其上没有固定探针，例如，可以是空载基底表面，即不带有探针的基底表面。所称的“基本一致”、“基本相同”同“一致”或“相同”，指不同批次制备、处理和/或平行试验产生的差异在允许的偏差范围内。
对第三探针在第三基底表面上的分布或排布方式不作限制，在本发明实施例中，第三探针在第三基底表面上随机分布或规则分布(例如呈阵列分布)。
在一个示例中，第一标记和第二标记为不同的荧光染料。在本发明的实施例中，检测第三基底表面上的信号，获得第三检测结果包括：通过成像系统对第三基底表面进行拍照，获得第三图像；以及检测第三图像，以获得第三检测结果；检测第四基底表面上的信号，获得第四检测结果包括：通过成像系统对第四基底表面进行拍照，获得第四图像；以及检测第四图像，以获得第四检测结果。成像系统可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机。
在本发明的实施例中，检测第三图像，以获得第三检测结果进一步包括：检测第三图像，以确定第三基底表面上只存在第三信号的位置的数量Nc3；检测第四图像，以获得第四检测结果进一步包括：检测第四图像，以确定第四基底表面上存在第三信号的位置的数量Nd。图像的检测可参考上述对第一图像的检测示例。
可利用公式(Nc3-Nd)/Nc3确定芯片表面固定有探针前后对待测核酸的非特异性吸附的变化，来实现基于第三检测结果和第四检测结果，检测待测核酸的非特异性吸附。
在一个例子中，如图4所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：加入末端带有Cy3荧光染料标记的探针(DNA捕获链-2，Capture DNA-2)，其序列与DNA杂交链不互补，探针为末端氨基化修饰后的 DNA单链，通过-NH 3与芯片表面修饰后带有的环氧基团反应，将DNA捕获链-2通过化学键连接到芯片表面；然后使用钝化液钝化芯片表面，以封闭未反应的环氧基团。
再加入杂交模板链，即待测核酸，模板链为末端带有Cy5荧光染料分子，进行杂交。杂交完成后，通过荧光显微镜包括全内反射荧光显微镜(TIRF)对表面进行拍照，可得到带有荧光信号的图像。(1)如果在同一个位置观察到两种荧光信号，则说明该位置有DNA双链同时存在，则为杂交模板链的链上特异性吸附，数量Nc1；(2)如果只观察到绿色荧光信号，则说明该位置只有捕获链而没有杂交链，数量Nc2；(3)如果只观察到红色荧光信号，则说明该位置只有杂交链，为杂交链在基底表面的非特异性吸附，数量Nc3。
如图5所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面，与上述基底表面相同，除了不包含探针，反应体系及反应时间与上述图4示例相同。
再加入杂交模板链，即待测核酸，进行杂交。杂交完成后，可通过荧光显微镜包括全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号的图像。这些荧光信号反映待测核酸在芯片表面的非特异性吸附，数量Nd。
然后，利用公式(Nc3-Nd)/Nc3评估确定基底表面固定探针前后对待测核酸的吸附变化。
在本发明的又一个实施例中，检测核酸特异性和/或非特异性吸附的方法包括：S10000：使待测核酸与第五探针反应，第五探针固定在第五基底表面上，待测核酸与第五探针至少部分互补，待测核酸带有第五标记，第五探针带有第六标记，第五标记能够产生第五信号，第六标记能够产生第六信号；S20000：检测第五基底表面上的信号，获得第五检测结果；S30000：使待测核酸与第六探针反应，第六探针固定在第六基底表面上，第六探针带有第六标记，待测核酸与第六探针不互补；S40000：检测第六基底表面上的信号，获得第六检测结果；S50000：使待测核酸与第七基底表面反应；S60000：检测第七基底表面上的信号，获得第七检测结果；S70000：基于第五检测结果、第六检测结果和第七检测结果中的至少两个检测结果，检测待测核酸的特异性和/或非特异性吸附。
利用该方法，能够定性或定量检测基底表面和/或基底表面上的探针对目标核酸的非特异和/或特异性吸附情况；通过检测系统检测基底表面的信号，能够定性或定量区分特异性和非特异性吸附，获得非特异性或特异性吸附的定量、分布情况等信息，能够用 于芯片生产以及所有涉及芯片检测核酸过程的方法或应用中，例如用于空载芯片(不带探针)或者捕获芯片(带探针)性能的评价、芯片生产的质控，用于芯片捕获核酸的效果的预测、分析比较等。
上述方法中，第五信号和第六信号是检测上可区分的信号，即能够检测到第五信号和第六信号是两个不同的信号，例如不同颜色的荧光信号等。
S10000、S30000和S50000的进行无顺序要求，可先后进行，也可同时进行。同样的，S20000、S40000和S60000的进行也无顺序限制。在一些实施例中，S10000、S30000和S50000是平行试验，为同时进行。
在一平行试验示例中，S10000、S30000和S50000中的第五基底、第六基底和第七基底的材质、表面特性如亲疏水、表面大小等是基本相同或一致的。在一个例子中，第五基底、第六基底和第七基底是经过相同表面处理的、相同材料性能的基底。在另一个例子中，第五基底、第六基底和第七基底是经过相同表面处理的同一基底的不同表面区域。例如，S10000和S30000中的第五基底表面和第六基底表面除了所固定的探针不一样，其它方面如表面大小、探针固定密度、探针分布、待测核酸的量、反应条件等基本一致。同时，S50000中的第七基底除了没有固定探针以外，其它方面如表面大小、待测核酸的量、反应条件等与S10000和S30000中的第五基底表面和第六基底基本一致。所称的“基本一致”、“基本相同”同“一致”或“相同”，指不同批次制备、处理和/或平行试验产生的差异在允许的偏差范围内。
对探针在基底表面上的分布或排布方式不作限制，在本发明实施例中，第五探针在第五基底表面上随机分布或规则分布(例如呈阵列分布)，类似地，第六探针在第六基底表面上随机分布或规则分布(例如呈阵列分布)。在平行实验中，第六探针在第六基底表面上的分布状态与第五探针在第五基底表面上的分布状态相同。
第五标记和第六标记可以为不同的荧光染料。在本发明的实施例中，检测第五基底表面上的信号，获得第五检测结果包括：通过成像系统对第五基底表面进行拍照，获得第五图像；以及检测第五图像，以获得第五检测结果。检测第六基底表面上的信号，获得第六检测结果包括：通过成像系统对第六基底表面进行拍照，获得第六图像；以及检测第六图像，以获得第六检测结果。检测第七基底表面上的信号，获得第七检测结果包括：通过成像系统对第七基底表面进行拍照，获得第七图像；以及检测第七图像，以获得第七检测结果。成像系统可以是带有成像系统的光学检测装置等，包括光源、物镜以及相机。
在本发明的实施例中，检测第五图像，以获得第五检测结果进一步包括：检测第五图像，以确定第五基底表面上同时存在第五信号和第六信号的位置的数量Ne1以及只存在第五信号的位置的数量Ne3。检测第六图像，以获得第六检测结果进一步包括：检测第六图像，以确定第六基底表面上同时存在第五信号和第六信号的位置的数量Nf1以及只存在第五信号的位置的数量Nf3。检测第七图像，以获得第七检测结果进一步包括：检测第七图像，以确定第七基底表面上存在第五信号的位置的数量Ng。图像的检测可参考上述对第一图像的检测示例。
进一步，进行以下(a)，(b)，(c)和(d)中的至少之一，来实现基于第五检测结果、第六检测结果和第七检测结果中的至少两个检测结果，检测待测核酸的特异性和/或非特异性吸附：(a)利用公式(Nf1+Nf3)/(Ne1+Ne3)确定待测核酸非特异性吸附在第五基底表面和第五探针上的比例；(b)利用公式Nf1/(Ne1+Ne3)确定待测核酸非特异性吸附在第五探针上的比例；(c)利用公式(Ne1+Ne3-Nf1-Nf3)/(Ne1+Ne3)确定待测核酸与第五探针特异性结合的比例；(d)利用公式(Nf3-Ng)/Nf3评估确定基底表面固定探针前后对待测核酸的吸附变化。
在一个例子中，如图3所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：加入末端带有Cy3荧光染料标记的探针(DNA捕获链-1，Capture DNA-1)，探针为经过氨基化修饰的探针、末端带有氨基基团，探针通过-NH 3与芯片表面的环氧基团反应，固定在基底表面，即将DNA捕获链-1通过化学键连接到芯片表面；然后使用钝化液钝化芯片表面，以封闭未反应的环氧基团。
加入模板链，即待测核酸，模板链为末端带有Cy5荧光染料分子并与DNA捕获链-1碱基互补配对的DNA杂交链(Target DNA)。杂交完成后，通过荧光显微镜例如全内反射荧光显微镜(TIRF)对表面进行拍照，可得到带有信号点/斑的图像。(1)如果同一个位置观察到两种荧光信号，则说明该位置有固定杂交的DNA双链或者存在部分少量的链上吸附，数量Ne1；(2)如果只观察到绿色荧光信号，则说明该位置只有捕获链而没有杂交链，数量Ne2；(3)如果只观察到红色荧光信号，则说明该位置只有杂交链，则为杂交链在基底表面的非特异性吸附以及极少量捕获链荧光猝灭的情况，数量Ne3。
如图4所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面：利用相同的反应体系及时间，将另一种探针固定在该表面上，该探针为带有Cy3荧光染料标记的DNA链(DNA捕获链-2，Capture DNA-2)，其序列与 DNA杂交链不互补。
加入与上述相同的杂交模板链，即待测核酸，进行杂交。杂交完成后，通过全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号点/斑的图像。(1)如果在同一个位置观察到两种荧光信号，则说明该位置有DNA双链同时存在，则为杂交模板链的链上特异性吸附，数量Nf1；(2)如果只观察到绿色荧光信号，则说明该位置只有捕获链而没有杂交链，数量Nf2；(3)如果只观察到红色荧光信号，则说明该位置只有杂交链，为杂交链在基底表面的非特异性吸附，数量Nf3。
如图5所示，在一基底表面上，该基底表面也称为芯片表面，为经过表面修饰后带有环氧基团的表面，与上述基底表面相同，除了不包含探针，反应体系及反应时间与上述图4示例相同。
再加入杂交模板链，即待测核酸，进行杂交。杂交完成后，可通过荧光显微镜包括全内反射荧光显微镜(TIRF)对表面进行成像拍照，可得到带有荧光信号的图像。这些荧光信号反映待测核酸在芯片表面的非特异性吸附，数量Ng。
基于上述信息，可进行量化区分特异性吸附和非特异性吸附，评估芯片表面和/或探针对待测核酸的非特异性吸附等，例如，利用公式(Nf1+Nf3)/(Ne1+Ne3)确定待测核酸非特异性吸附在基底表面和探针上的比例；利用公式Nf1/(Ne1+Ne3)确定待测核酸非特异性吸附在探针上的比例；利用公式(Ne1+Ne3-Nf1-Nf3)/(Ne1+Ne3)确定待测核酸与探针特异性结合的比例；利用公式(Nf3-Ng)/Nf3评估确定基底表面固定探针前后对待测核酸的吸附变化。
以下通过实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。涉及的材料、试剂以及序列等，如无特殊说明，可通过自己配制合成或者市售途径获取。
实施例1：考察DNA杂交过程中的非特异性吸附的影响
芯片：表面带有环氧基团的玻璃(购自肖特)；
固定链1(EKB-6P-Cy3)：人工合成的特定序列，末端带有-NH 3以及荧光基团Cy3染料的DNA短序列，序列具体为：
TTTTTTTTTTTTACTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAG-3’(SEQ ID NO：1)；
固定链2(EKB-7P-Cy3)：人工合成的特定序列，末端带有-NH 3以及荧光基团Cy3染料的DNA短序列，序列具体为：
TTTTTTTTTTTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTAC-3’(SEQ ID NO：2)；
杂交链(EKB-6T-Cy5)：人工合成的与固定链1互补配对，与固定链2不互补配对，带有长度为35bp待测序列，末端带有荧光基团Cy5的DNA短序列，序列具体为：
5’-GATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAA TACCATGCAGAAGGAGGCAAAGTA-3’(SEQ ID NO：3)。
实验过程：
实验组：将玻璃清洗吹干后，置于150mMK 2HPO 4并含有浓度为1.0M的氨基修饰的固定链1(EKB-6P-Cy3)核酸探针的溶液中，37℃条件下反应0.5小时，依次用3XSSC溶液(含0.1％的Triton)，3XSSC，0.15M K 2HPO 4溶液清洗后，加入1M K 2HPO 4在37℃条件下钝化17小时。
对照组1：将玻璃清洗吹干后，置于150mM K 2HPO 4并含有浓度为1.0M的氨基修饰的固定链2(EKB-7P-Cy3)核酸探针的溶液中，37℃条件下反应0.5小时，依次用3XSSC溶液(含0.1％的Triton)，3XSSC，0.15M K 2HPO 4溶液清洗后，加入1M K 2HPO 4在37℃条件下钝化17小时。
对照组2：将玻璃清洗吹干后，置于150mM K 2HPO 4溶液中，37℃条件下反应0.5小时，依次用3XSSC溶液(含0.1％的Triton)，3XSSC，0.15M K 2HPO 4溶液清洗后，加入1M K 2HPO 4在37℃条件下钝化17小时。
芯片(Flow Cell，流通池)组装：将钝化后的玻璃与其它基片或基底组装成多通道的芯片。
杂交链杂交：将组装好的多通道流通池加入Rinse buffer(1XSSC+150mM HEPES+0.1％SDS)，在55℃条件下复溶0.5小时，然后再加入杂交链(EKB-6T-Cy5)浓度为1nM的3XSSC溶液，在55℃条件下反应0.5小时，然后依次使用Rinse buffer(1XSSC+150mM HEPES+0.1％SDS)和Buffer H(150mM HEPES+150mM NaCl)冲洗通道。
拍照检测：使用TIRF同时对芯片表面进行拍照。
利用上述示例的方法对图像进行检测，包括对亮点/亮斑进行识别、定位和计数，获得结果如下：
实验组结果如图6所示，其中左图显示一个视野中的Cy3荧光点，右图显示相同视野中的Cy5荧光点，重合比例大约80％。
对照组1结果如图7所示，其中左图显示一个视野中的Cy3荧光点，右图显示相同视野中的Cy5荧光点，其中重合比例为。
对照组2结果如图8所示，该图显示一个视野中的Cy5荧光点，该实验中的Cy5荧光点为杂交链在芯片表面的非特异性吸附。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (10)

  1. 一种检测核酸特异性和/或非特异性吸附的方法，其特征在于，所述方法包括：
    使待测核酸与第一探针反应，所述第一探针固定在第一基底表面上，所述待测核酸与所述第一探针至少部分互补，所述待测核酸带有第一标记，所述第一探针带有第二标记，所述第一标记能够产生第一信号，所述第二标记能够产生第二信号；
    检测所述第一基底表面上的信号，获得第一检测结果；
    使所述待测核酸与第二探针反应，所述第二探针固定在第二基底表面上，所述第二探针带有所述第二标记，所述待测核酸与所述第二探针不互补；
    检测所述第二基底表面上的信号，获得第二检测结果；
    基于所述第一检测结果和所述第二检测结果，检测所述待测核酸的特异性和/或非特异性吸附。
  2. 一种检测核酸非特异性吸附的方法，其特征在于，所述方法包括：
    使待测核酸与第三探针反应，所述第三探针固定在第三基底表面上，所述待测核酸带有第三标记，所述第三探针带有第四标记，所述待测核酸与所述第三探针不互补，所述第三标记能够产生第三信号，所述第四标记能够产生第四信号；
    检测所述第三基底表面上的信号，获得第三检测结果；
    使待测核酸与第四基底表面反应；
    检测所述第四基底表面上的信号，获得第四检测结果；
    基于所述第三检测结果和所述第四检测结果，检测所述待测核酸的非特异性吸附。
  3. 根据权利要求1或2所述的方法，其特征在于，所述第一探针在所述第一基底表面上的分布状态选自随机分布和规则分布中的至少一种，所述第二探针在所述第二基底表面上的分布状态与所述第一探针在所述第一基底表面上的分布状态相同；和/或
    所述第三探针在所述第三基底表面上的分布状态选自随机分布和规则分布中的至少一种。
  4. 根据权利要求1或2所述的方法，其特征在于，
    (i)所述检测所述第一基底表面上的信号，获得第一检测结果包括：通过成像系统对所述第一基底表面进行拍照，获得第一图像；以及检测所述第一图像，以获得所述第一检测结果；
    所述检测所述第二基底表面上的信号，获得第二检测结果包括：通过所述成像系统对所述第二基底表面进行拍照，获得第二图像；以及检测所述第二图像，以获得所述第 二检测结果；和/或
    (ii)所述检测所述第三基底表面上的信号，获得第三检测结果包括：通过成像系统对所述第三基底表面进行拍照，获得第三图像；以及检测所述第三图像，以获得所述第三检测结果；
    所述检测所述第四基底表面上的信号，获得第四检测结果包括：通过所述成像系统对所述第四基底表面进行拍照，获得第四图像；以及检测所述第四图像，以获得所述第四检测结果。
  5. 根据权利要求4所述的方法，其特征在于，
    (i)中检测所述第一图像，以获得所述第一检测结果包括：检测所述第一图像，以确定所述第一基底表面上同时存在第一信号和第二信号的位置的数量Na1以及只存在第一信号的位置的数量Na3；
    检测所述第二图像，以获得所述第二检测结果包括：检测所述第二图像，以确定所述第二基底表面上同时存在第一信号和第二信号的位置的数量Nb1以及只存在第一信号的位置的数量Nb3；和/或
    (ii)中检测所述第三图像，以获得所述第三检测结果包括：检测所述第三图像，以确定所述第三基底表面上只存在第三信号的位置的数量Nc3；
    检测所述第四图像，以获得所述第四检测结果包括：检测所述第四图像，以确定所述第四基底表面上存在第三信号的位置的数量Nd。
  6. 根据权利要求5所述的方法，其特征在于，
    进行(i)后，所述基于所述第一检测结果和所述第二检测结果，检测所述待测核酸的特异性和/或非特异性吸附包括进行以下(a)，(b)和(c)中的至少之一：
    (a)利用公式(Nb1+Nb3)/(Na1+Na3)确定所述待测核酸非特异性吸附在所述第一基底表面和所述第一探针上的比例；
    (b)利用公式Nb1/(Na1+Na3)确定所述待测核酸非特异性吸附在所述第一探针上的比例；
    (c)利用公式(Na1+Na3-Nb1-Nb3)/(Na1+Na3)确定所述待测核酸与所述第一探针特异性结合的比例；和/或
    进行(ii)后，所述基于所述第三检测结果和所述第四检测结果，检测所述待测核酸的非特异性吸附包括：利用公式(Nc3-Nd)/Nc3确定芯片表面固定有探针前后对所述待测核酸的非特异性吸附的变化。
  7. 根据权利要求4或5所述的方法，其特征在于，
    (i)中，所述第一图像和/或所述第二图像包含多个像素点，检测所述第一图像和/或检测所述第二图像包括：
    利用k*k矩阵对所述第一图像和/或所述第二图像进行亮点检测，包括判定所述矩阵的中心像素值不小于该矩阵非中心任一像素值的矩阵对应一个亮点，k为大于1的奇数，k*k矩阵包含k*k个像素点；
    任选的，所述矩阵的中心像素值大于第一预设值，所述矩阵非中心任一像素值大于第二预设值；
    任选的，所述第一预设值和所述第二预设值与该图像的平均像素值相关；和/或
    (ii)中，所述第三图像和/或所述第四图像包含多个像素，检测所述第三图像和/或检测所述第四图像包括：
    利用k*k矩阵对所述第三图像和/或所述第四图像进行亮点检测，包括判定所述矩阵中心的像素值不小于该矩阵非中心任一像素值的矩阵对应一个亮点，k为大于1的奇数，k*k矩阵包含k*k个像素点；
    任选的，所述矩阵中心的像素值大于第一预设值，所述矩阵非中心任一像素值大于第二预设值；
    任选的，所述第一预设值和所述第二预设值与该图像的平均像素值相关。
  8. 根据权利要求4或5所述的方法，其特征在于，
    (i)中，所述第一图像和/或所述第二图像包含多个像素点，检测所述第一图像和/或检测所述第二图像包括：
    亮点检测步骤，所述亮点检测步骤包括：分析所述第一图像和/或分析所述第二图像以计算亮点判定阈值，分析所述第一图像和/或所述第二图像以获取候选亮点，根据所述亮点判定阈值判断所述候选亮点是否为所述亮点，和/或
    (ii)中，所述第三图像和/或所述第四图像包含多个像素点，检测所述第三图像和/或检测所述第四图像包括：
    亮点检测步骤，所述亮点检测步骤包括：分析所述第三图像和/或分析所述第四图像以计算亮点判定阈值，分析所述第三图像和/或所述第四图像以获取候选亮点，根据所述亮点判定阈值判断所述候选亮点是否为所述亮点。
  9. 根据权利要求8所述的方法，其特征在于，(i)和/或(ii)的亮点检测步骤之前还包括：
    图像预处理步骤，所述图像预处理步骤包括预处理所述第一图像、第二图像、第三图像和/或第四图像，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    所述图像预处理步骤包括进行以下(a)、(b)、(c)、(d)、(e)和(f)至少之一：
    (a)对所述第一图像、第二图像、第三图像和/或第四图像进行减背景处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (b)对进行减背景处理后的第一图像、减背景处理后的第二图像、减背景处理后的第三图像和/或减背景处理后的第四图像进行简化处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (c)对所述第一图像、第二图像、第三图像和/或所述第二图像进行滤波处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (d)对所述第一图像、第二图像、第三图像和/或第四图像进行减背景处理后再进行滤波处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (e)对进行减背景处理后再进行滤波处理的第一图像、进行减背景处理后再进行滤波处理的第二图像、进行减背景处理后再进行滤波处理的第三图像和/或进行减背景处理后再进行滤波处理的第四图像进行简化处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像；
    (f)对所述第一图像、第二图像、第三图像和/或第四图像进行简化处理，以获得预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像。
  10. 根据权利要求9所述的(b)、(e)和(f)任一方法，其特征在于，所述根据所述亮点判定阈值判断所述候选亮点是否为所述亮点，包括：在所述预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像中查找大于(p*p-1)连通的像素点并将查找到的所述像素点作为所述候选亮点的中心，p为自然数且为大于1的奇数；
    判断所述候选亮点的中心是否满足条件：I max*A BI*ceof guass＞T，其中，I max为p*p 窗口的中心最强强度，A BI为p*p窗口中所述预处理后的第一图像、预处理后的第二图像、预处理后的第三图像和/或预处理后的第四图像中为设定值所占的比率，ceof guass为p*p窗口的像素和二维高斯分布的相关系数，T为所述亮点判定阈值，
    若满足上述条件，判定所述候选亮点为一个亮点。
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