CN100347545C - Method of preoceeding qualitative and/or quantitative analysis against target substance in sample and its detecting device - Google Patents
Method of preoceeding qualitative and/or quantitative analysis against target substance in sample and its detecting device Download PDFInfo
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Abstract
The present invention relates to a method and a device for qualitatively and/or quantitatively analyizing target substances in samples, particularly to biological samples, by using a colored biological chip. Due to the utilization of the obvious contrast of signal light color codes or/and saturation or/and brightness between a colored background and a colored target to make a preferred scheme form the maximum contrast, the analysis sensitivity is enhanced, the freedom degrees of chip base selection and scanner selection are greatly widened, and high detection sensitivity can be realized under the condition of low detection cost.
Description
Technical field
The present invention relates to a kind of in the sample, the detection method and the pick-up unit thereof that carry out qualitative and/or quantitative test of the object in the biological sample particularly.
Background technology
To in the sample, the method for carrying out qualitative and/or quantitative test of the object in the biological sample particularly, its wide range, wherein the ultimate principle based on a class of probe-object specific reaction is: make the target molecule in probe and the sample carry out idiosyncrasy, analyze the result of this idiosyncrasy again.The basic step of this type of analytical approach is: prepare a kind of device that contains the probe reaction device, sample contacted go forward side by side the line correlation reaction with described reactor, analyze described reaction result.Because the difference of used probe reaction apparatus, derive the method for a lot of qualitative and/or quantitative test, for example, biochip method, PCR method, quick detection reagent bar method, ELISA method, immunofluorescence technique, or the like.
In the present invention, " pick-up unit " in the qualitative and/or quantitative analysis method, be meant the relevant apparatus that includes reactor in qualitative and/or the quantitative analysis method, wherein be fixed with in the reactor in order to probe with the target molecule generation idiosyncrasy in the sample, biological example chip or biochip kit, quick detection reagent bar or quick detection kit, ELISA Plate or ELISA Plate kit, or the like; " biochip " is meant a kind of pick-up unit in qualitative and/or the quantitative analysis method, and its reactor middle probe can be discerned in addressable mode with the result of the target molecule generation idiosyncrasy in the sample; " biochip method " be meant utilize biochip in the sample, the method for carrying out qualitative and/or quantitative test of the object in the biological sample particularly.
The most frequently used biochip is polypeptide chip and genetic chip.Polypeptide chip is the biochip for preparing on substrate as probe stationary with a plurality of amino acid whose sequential structures (comprising protein).Genetic chip is with sample amplifying nucleic acid to be checked, nucleotide and complementary nucleic acid, nucleotide probe hybridization, forms crossbred, or combines with specific antibody, shows the chip of testing result again with color reaction.The biochip scope that has a wide range of applications comprises gene expression detection, genescreen, drug screening, medical diagnosis on disease treatment, environmental monitoring and fields such as improvement, judicial expertise.The core of biochip is wherein fixing probe.The probe of biochip comprises that all can be fixed on the material of the biologically active on the solid phase carrier, for example antigen, antibody, strand and multichain DNA, RNA, nucleotide, part, aglucon, polypeptide, cell, be organized into the biotic component that grades.
The existing problem that waits to solve based on the qualitative and/or quantitative analysis method and the pick-up unit of biochip is described below simply.
The prior biological chip method comprises luminous detection method and non-luminous survey method two classes of picking up.The non-luminous example of picking up the survey method has SELDI-TOF-MS method (surperficial laser enhanced is dissociated and mass spectrum Surface-Enhanced Laser Desorption/Ionization-Time Of Flight-MassSpectra during the flying of laser ionization, and for example comprises the ProteinChip Array system of the U.S. Ciphergen company of sheet metal base).Yet the biochip test method of at present widespread use still is a luminous detection method.Luminous detection method mainly comprises fluorescence detection, chemiluminescence detecting method and complex light irradiating and detecting method.At present, in the fluorescence detection used sheet base be basically diascope (for example, amination, aldehyde radicalization, poly-D-lysine sheet base), used base is transparent slide, plastic plate or metal film (for example silver-colored film) basically in the chemiluminescence detecting method, the used diffusible basically polymer film of base (for example pvdf membrane, nylon membrane, nitrocellulose membrane, cellulose acetate film etc.) in the complex light irradiating and detecting method.Yet based on the complex light irradiating and detecting method of diffusible polymer membrane base, its sensitivity is not high at present; Present chemiluminescence detecting method sensitivity is also not really high; Present fluorescence detection, though the above two height of remolding sensitivity, but still exist the ground unrest of slide sheet base not low, weak points such as activation sheet base cost height, detecting instrument costliness, influenced the large-scale application of biochip method.In a word, present biochip method and biochip wait to improve sensitivity and reduce the detection cost.
Summary of the invention
The present invention does not reduce its sensitivity even improves its sensitivity with the cost that reduces biochip luminous detection method is main target.Thereby, research of the present invention selected " maximize with the optics contrast of colorant and sheet base and guarantee or improve detection sensitivity; reduce the technical requirement of sheet base and enlarge sheet base range of choice, thereby and the technical requirement that reduces the luminous detection instrument enlarge the instrument range of choice " technology path realize this target.
In fact, target of the present invention is relevant with the target of present stealth technology, and stealth technology efforts be made so that target and background have minimized contrast, and technology of the present invention efforts be made so that target and background have maximized contrast.Technology of the present invention can be described as the body technology that shows, and detection method of the present invention can be described as and shows the body technology detecting method.The certain methods of stealth technology even some materials of using (for example blacker-than-black material etc.) are useful especially for the purpose that reaches us.For example, for infrared stealth technology, the size of human contrast emitance C such as Maclean is represented the detectivity of thermal imaging system: C=Eo-Eb.Eo is the target emissivity in the formula, and Eb is a background emitance rate, and Eo and Eb have proportional relation with the absorptivity of target material and background material respectively.For infrared stealth, C is big more, and thermal imaging system resolution is high more, detectivity is big more.Otherwise, be in stealthy optimum condition when C goes to zero.In like manner, the sensitivity of biochip method also is relevant with the contrast of the optical property of target (for example colorant) material and background (sheet base) material.In our research, we find, use the big sheet base-colorant system of contrast, can improve the detectivity of object-example reaction, thereby introduce more biochip or biochip kit newtype, increase the selection degree of freedom of biochip test method, to reach target of the present invention (seeing embodiment).
Thereby, the invention provides a kind of method that object in sample, the particularly biological sample is carried out qualitative and/or quantitative test, it comprises following basic step:
A, object or the material that contains object are contacted with the reactor of pigmented biological chip and reaction therein, described have the color chip reactor to contain to provide the colored pellets of a coloured background base, the probe in the described reactor on the colored pellets base or the density of probe-particulate greater than 2 points/cm2, preferred version greater than 10 points/cm2, more preferably scheme is greater than 20 points/cm2;
B, colorant or the material that contains colorant are anchored at reaction place in the described reactor of a, to be formed with Semu mark;
C, with the monochromatic or compound irradiation light of wavelength between 200-780nm, maybe other luminous condition of colorant is applied in the described reactor of b;
D, to light, coloured background and coloured target in the described reactor of c and the signal light that also has the interaction between the signal detection device to produce sometimes detect;
E, according to the described described reaction result of interpretation of result a that detects of d.
In the present invention, " chip reactor " or abbreviation " reactor " are meant and are fixed with probe array in the biochip, when detecting, reach other dependency structure that is communicated with it with the place of object generation specific reaction, " sheet base " is meant the solid phase carrier in order to stationary probe or probe-particulate, " probe " is meant and is fixed in the reactor with object generation selective reaction object is carried out the material of qualitative and/or quantitative test, " probe-particulate " is meant the micron or the carrier of nano-scale that is fixed with probe, and " point " in the probe density unit is meant probe or/and probe-particulate is fixed on a diacritic zone that the border is arranged that forms on the sheet base.
In the present invention, " colored pellets base " is meant the non-porous diaphragm base that opaque, no minute surface is reflective.Colored pellets base among the present invention also independently provides coloured background except that as the solid phase carrier, thereby is different from widely used inorganic or organic filmbase, reflective tinsel sheet base and the too high porous membrane base of surfaceness of minute surface now.In the present invention, " colorant " be meant can with the reaction result of object in reactor with the color code of the light of wavelength between 200-780nm or/and saturation degree or/and the mode of brightness is carried out the mark substance of mark, " autonomous luminescent substance " is meant the material that does not need the outside that irradiation light is provided and produced signal light by chemistry or electrochemical reaction.
Another aspect, the method for qualitative and/or quantitative test provided by the invention, in described interaction, form color code between wherein coloured background and the coloured target or/and saturation degree or/and the obvious contrast of brightness, preferred version form the maximization contrast.
In the present invention, " color code " is meant the different colouring discriminations that form of wavelength (predominant wavelength) that the accounting example is the highest in the spectral reflectance, " saturation degree " is meant the purity (contain colour killing number percent heal big color more unsaturated) of color, and " brightness " is meant the bright degree of color.
On the other hand, qualitative and/or quantitative analysis method provided by the invention, when the main signal light that provides by colorant on described coloured target wherein, described coloured background of base to the light reflectivity of signal light less than 10%.Provide signal light on described coloured target by colorant, be meant that colorant is from main light emission (for example autonomous chemiluminescence), or luminous under the irradiation light effect (for example colorant is a fluorescent material), or luminous under other external effect (for example electrochemiluminescence), or the like.In these cases, the light reflectivity of coloured background is more little, and the observability of the coloured target on then coloured background is high more.
Qualitative and/or quantitative analysis method provided by the invention, the material (for example fluorescent material and chemiluminescent substance) of colorant for the signal light on described coloured target can be provided wherein, the sheet base is the sheet base of black for dark (for example peony, bottle green, or the like), preferred version for its coloured background under white light.
Another aspect, qualitative and/or quantitative analysis method provided by the invention, when wherein mainly providing signal light on described coloured target by the selective reflecting of colorant, described coloured background of base to the selective reflecting rate of signal light less than 10% or its non-selective reflectivity greater than 50%.In the case, the selective reflecting rate of colorant is big more, the more little or non-selective reflectivity of the selective reflecting rate of coloured background is big more, and the observability of the coloured target on then coloured background is high more.
Qualitative and/or quantitative analysis method provided by the invention, wherein said colorant are monochromatic or approaching monochromatic colorant, and preferred version is that predominant wavelength is the colorant of red, green, blueness, cyan or purple under white light; Described base is that its coloured background is white sheet base for light color, preferred version under white light.
Another aspect, qualitative and/or quantitative analysis method provided by the invention, wherein when mainly providing signal light on described coloured target by the non-selective reflection of colorant, the non-selective reflectivity of the coloured background of described substrate is greater than 50% or less than 10%.In the case, the non-selective reflectivity of colorant is big more, the non-selective reflectivity of coloured background is more little, perhaps the non-selective reflectivity of colorant non-selective reflectivity more little, coloured background is big more, and the observability of the coloured target on then coloured background is high more.
Qualitative and/or quantitative analysis method provided by the invention, wherein said colorant is black or dark colorant, and described base be that its coloured background is white sheet base for light color (for example pale yellow color chips base, light gray color chips base, or the like), preferred version under white light; Perhaps described colorant is light color or white colorant, and described base is the sheet base of black for dark (for example peony, bottle green, or the like), preferred version for its coloured background under white light.Be characterised in that: described colorant is black or dark color (for example peony, bottle green, or the like), and described coloured background of base is that light color (for example pale yellow color chips base, light gray color chips base, or the like), preferred version are white under white light.
Another aspect, the invention provides a kind of biochip, it is characterized in that: include color chips base and probe in its reactor at least or/and the probe particulate, the probe in the described reactor on the colored pellets base or the density of probe-particulate greater than 2 points/cm2, preferred version greater than 10 points/cm2, more preferably scheme is greater than 20 points/cm2.Described colored pellets base is identical with above-mentioned colored pellets base in the qualitative and/or quantitative analysis method provided by the invention.
Biochip provided by the invention, its colored pellets base comprise monobasic colored materials sheet base, colored coating-structure sheet sheet base, coloured diaphragm-structure sheet sheet base, minimum reflected structural sheet-coloured structure sheet sheet base, see through-colored materials sheet base and nanometer colored materials sheet base.
In the present invention, " monobasic colored materials sheet base " is meant by the sheet base that forms described coloured background and sheet based structures with a kind of colored materials, " colored coating-structure sheet sheet base " is meant by colored coating and forms described coloured background and other material forms the sheet base of sheet base geometry, " coloured diaphragm-structure sheet sheet base " is meant by coloured diaphragm and forms described coloured background and other material forms the sheet base of sheet base geometry, " minimum reflected structural sheet-coloured structure sheet sheet base " is meant the sheet base that is formed described coloured background by reflection structure layer and coloured structure sheet, " see through-colored materials sheet base " and be meant that " nanometer colored materials sheet base " is meant the sheet base that is formed described coloured background by the nanometer colored materials by transparent or filter and the common sheet base that forms described coloured background of colored materials.
Biochip provided by the invention, it is characterized in that: form in its sheet base in colored materials, transparent material and the filter of described coloured background, contain following one or more organism: nitrocellulose, polystyrene, polyacrylate, polysulfones, polyethersulfone, Polyvinylchloride, amino resins, or the like.In fact, all conform to above-mentioned " developing technology " principle, directly linking probe or indirect linking probe (for example connecting medium by particulate or the poly-polysaccharide of modification) and the coloring matter of not losing the probe activity all can be manufactured with the color chips base.
Biochip provided by the invention, in its nanometer colored materials sheet base, described nanoparticle mean grain size is 1-500nm, be distributed in sheet base part or all surfaces or be distributed in the colored materials layer.In fact, nanoparticle in colored pellets base of the present invention, color code between may command colored pellets base and the colorant or/and saturation degree or/and luminance contrast, and/or sheet base end face surface hydrophilicity.A lot of documents prove, and nanoparticle can change the optical property (for example light transmission, absorptivity, surface gloss, or the like) of material.Nano material is used for " stealth technology " the sixth of the twelve Earthly Branches.Another aspect, the nanoparticle that adds possess hydrophilic property can be improved the hydrophobicity (some colored paint sheet base often has hydrophobic surface) of hydrophobic surface again, and suitable sheet base water wettability is one of important control basis of reactivity such as biochip stability.
Biochip provided by the invention, nanoparticle in its nanometer colored materials sheet base, comprise gold, vanadium, lead, silver, iron and oxide fine particle thereof, monox, titanium dioxide, alumina particulate, plastics, polysaccharide, latex, resin and other macromolecular material particulate comprise that also above-mentioned particulate modification is combined with the particulate derivant of functional group and/or function thing.
Another aspect, biochip provided by the invention, its probe comprises part, aglucon, antigen, antibody, polypeptide, protein and strand or multichain DNA, RNA, nucleotide, and its probe-particulate is that mean grain size is of a size of 0.1nm-10um, preferred version is the particulate of 1-100nm and the connector of probe.
Another aspect, biochip provided by the invention, stationary probe on its colored pellets base is or/and probe particulate place and peripheral region thereof, under white light, look it is following a kind of color: black, predominant wavelength is the dark color of redness, green, blueness, cyan or purple, white, and predominant wavelength is the light color of redness, yellow, green, blueness, cyan or purple.
On the other hand, biochip provided by the invention is characterized in that: provide on the described colored pellets base coloured background the surface roughness Ra between the 0.02-3.0um, preferred version is between 0.25-3.0um.In some cases, surface smoothness too high do not help improving between described coloured background and the coloured target color code or/and saturation degree or/and the contrast of brightness.
Another aspect the invention provides a kind of biochip kit, it is characterized in that: the material that it contains pigmented biological chip and colorant or contains colorant.Pigmented biological chip in the biochip kit of the present invention is same as above-mentioned pigmented biological chip.Colorant in the biochip kit of the present invention or contain the material of colorant is same as above-mentioned colorant in the qualitative and/or quantitative analysis method provided by the invention or contains the material of colorant.
Biochip kit provided by the invention, its colorant comprises fluorescent material, chemiluminescence catalyzer, colour developing and luminous substrate, non-ferrous metal or the non-ferrous metal salt that is used as the biochip mark substance at present, and the organic and inorganic dyestuff, the pigment that are not used as the biochip mark substance at present as yet; Its material that contains colorant comprises colored marker, nanoparticle colored marker and molecular vehicle colored marker.
In the present invention, " colored marker " is meant the label that contains colorant and mark aglucon, " nanoparticle colored marker " is meant and contains particulate colorant and mark aglucon, that be used as the nano-scale of label, and " molecular vehicle colored marker " is meant the label that contains colorant, mark aglucon and their molecular vehicle.The use of nanoparticle colored marker among the present invention makes the range of choice of colorant enlarge.In fact, be combined with functionalization two, six family's semi-conductor nano particles of antibody or DNA, be combined with the rare earth nano particulate of antibody, can be in conjunction with the organism bag of the different colours of biological substance by nano particle, or the like the existing report of new label, can be with in the methods of the invention.
Biochip kit provided by the invention, the nanoparticle in its nanoparticle colored marker is for fixedly aglucon and/or colorant do not lose the nanoparticle of its activity, mean grain size 1-500nm.
Biochip kit provided by the invention is characterized in that: the particulate in its contained nanoparticle colored marker comprises gold, vanadium, lead, silver, iron and oxide fine particle thereof; Monox, titanium dioxide, alumina particulate; Plastics (for example: polystyrene type, polyvinyl chloride, PP type, polyacrylamide, polyimide, polyethers ketone), polysaccharide (for example: glucosan, agarose, starch, and their modifier), (for example: particulate nylon) comprises that also above-mentioned particulate modifies the particulate derivant that is combined with functional group and/or function thing for latex, resin and other macromolecular material.In fact, fixedly aglucon and/or colorant and not lose its bioactive particulate a lot, for example be used for biochemical and immunologic detection method Nano microsphere, be used as some nano-carrier of slow releasing carrier of medication, or the like.
Biochip kit provided by the invention is characterized in that: described functional group comprises amino, acyl group, carboxyl, hydroxyl, aldehyde radical and epoxy radicals, or the like.
The present invention also provides the preparation method of a kind of analyzing biochips device or biochip kit, and its concrete steps are:
A, provide above-mentioned colored pellets base, probe or/and probe-particulate, make the color chip that has of described coloured background;
B, provide above-mentioned colorant, aglucon or/and nano particle is made described coloured label or/and coloured marking nano particulate.
The advantage of analyzing biochips method and apparatus of the present invention is: with regard to biochip, compare with the filmbase that accounts for main flow at present, the supply scope of the coloured probe card among the present invention is very big, for the different needs of different analyzing and testing provides very big degree of freedom, provide much higher feasibility for reducing sheet base cost; With regard to Mk system because can the wide colorant of usable range, for utilizing more easily scanner to detect to maximize color code between coloured background and the coloured target or/and saturation degree or/and luminance contrast provides high selection degree of freedom; In a word, analyzing biochips method of the present invention can realize higher detection sensitivity low the detection under the cost condition.
Specific embodiment
Embodiment 1: fluorescence shows body detection method and black biochip
In this example, used biochip is the black biochip that contains the black patch base, used colorant is a rhodamine, the goat-anti people two anti-(U.S. JacksonImmunoRresearch Laboratories company) that the used label that contains colorant is the rhodamine mark, used ginseng photo-based is the transparent amino modified slide (U.S. Telechem International company, below note is made sheet base 0) of size 75 * 25 * 1mm.
The present embodiment step is as follows:
The preparation of-black patch base:
The prepared black background sheet base of present embodiment is: monobasic black material sheet base, black coating-structure sheet sheet base, black diaphragm-structure sheet sheet base and transparent-black material composite sheet base.
The prepared monobasic black material sheet base of present embodiment is a black plastic sheet base, and it is the thermoplastic polyvinyl chloride after adding filling agent such as channel black, makes through compression molding, is of a size of 75 * 25 * 1mm, and below note is made sheet base 1.
Present embodiment coatings prepared-structure sheet sheet base is a black organic coating sheet base, and coating is formed by self-control black high polymeric solution, and the structure sheet is the transparent microslide (U.S. Erie Scientific company) of size 75 * 25 * 1mm.The used black high polymeric solution of present embodiment is for being colorant with channel black (Lu thirty natural gas chemical plant), being solvent with benzene, containing the Polyvinylchloride dark solution of an amount of tackifier (tackifier 555, Chengdu morning twilight chemical design institute).The Polyvinylchloride dark solution is sprayed on the microslide, and dry back forms black coating.Formed black film layer thickness is about 30um.Although in coating-structure sheet sheet base, on the end face of structure sheet and bottom surface, can form black coating, only on the end face of structure sheet, form black coating in the present embodiment.More than the black multicomponent material sheet base of preparation note is made sheet base 2.
Black diaphragm-structure sheet sheet base that present embodiment is prepared, its coloured diaphragm is the black pvc film (thickness 80um) that is cut into planar dimension 75 * 25mm behind the compression molding, and its structure sheet is the transparent microslide (Erie Scientific company) of 75 * 25 * 1.0mm.The black pvc film is combined in end face on the transparent microslide by thermal bonding process.Prepared base note made sheet base 3.
Transparent-black material composite sheet base that present embodiment is prepared, its transparent material are the polyvinyl chloride film of 75mm * 25mm * 15um (long * wide * thick), and its colored materials is the black plastic sheet base of above-mentioned preparation.The polyvinyl chloride film thermoplastic is fitted in the end face of black plastic sheet base.The sheet base note that makes is made sheet base 4.
The preparation of-black biochip:
The black biochip of present embodiment is to lead on the end face of above-mentioned black patch base to form the reactor isolation structure and make sheet Ji Chi, then at sheet base pond internal fixation probe and the sheet base that exposes in confining liquid closure plate base pond form.
The isolation structure of biochip reaction device, be to spread upon on the 4 kinds of sheet bases and ginseng photo-based (sheet base 0) of above-mentioned preparation with high hydrophobic organosilicon coating (Chengdu morning twilight chemical design institute), drying and forming-film (thickness is less than 0.05mm) back (with reference to our another invention: " a kind of biochip and preparation method of reactor isolation structure minimized height ", number of patent application 03117397.7) of on the sheet base, forming then.Per 1 sheet base forms 8 sheet Ji Chi, and each sheet base pool size is 4.5mm * 4.5mm, and sheet base cell compartment is 4.5mm from structure width.Sheet base pond isolation structure on the sheet base 0 directly is formed on the end face of amination slide, the reactor isolation structure of sheet base 1 directly is formed on the black plastic sheet base end face, reactor isolation structure on the sheet base 2 directly is formed on the black coating surface, sheet base top, and the reactor isolation structure on the sheet base 3 directly is formed on the black pvc film surface of sheet base top, the reactor isolation structure on the sheet base 4 directly is formed on the sheet base top transparent film surface.
The used probe of present embodiment is HCV antigen (the People's Hospital, BeiJing, China hepatopathy research institute) and HIVI+2 antigen (the People's Hospital, BeiJing, China hepatopathy research institute).In the sheet base pond of each above-mentioned preparation, respectively with HCV antigen (1.5mg/ml) and the fixing reactor that forms of HIV1+2 antigenic solution (1.5mg/ml) point sample.In a reactor, 2 diameters of two kinds of antigen each points are the point of 200um, form 2 * 2 arrays.After bag is done, standby with the confining liquid sealing.Prepared chip, according to employed base numbering, note is done chip 0, chip 1, chip 2, chip 3, chip 4 respectively.
-fluorescence shows the body detection method:
In the present embodiment, No. 1 sample is a HCV antibody positive serum, No. 2 samples are HIV1+2 antibody positive human serum, No. 3 positive testers of sample (potpourri of HCV antibody and HIV1+2 antibody positive serum tester), No. 4 negative testers of sample (the serum tester that HCV antibody and HIV1+2 antibody are all negative).All samples are all through using classical ELISA method to detect in advance under 20 times of diluting reaction conditions of serum.
4 kinds of samples add respectively in the reactor of said chip during experiment.In 4 reactors of each biochip, be added with the sample of dilution in 1: 50 respectively.The application of sample amount is 5ul.React after 30 minutes and wash 5 times, the each addition of cleansing solution is 15ul.
Used colorant is a rhodamine, and label is the goat-anti people two anti-(JacksonImmunoRresearch Laboratories company) of rhodamine mark, and addition is 5ul, reaction back washing 5 times, and the each addition of cleansing solution is 15ul, dry laggard line scanning.Scanner is confocal laser scanner (GMS of Afymetrix company 418 chip scanners), scanning excitation wavelength 532nm, wavelength of transmitted light 570nm, the treated software of the signal that reads (Afymetrix JAGUAR 2.0) is handled, and obtains result such as table 1 after averaging then.
Table 1 fluorescence shows the testing result of body biochip
| Chip | Sheet base reflectivity (%) | HCV antibody positive serum | HIV antibody positive serum | Positive control | The negative control thing | ||||
| HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | ||
| Chip 0 | Do not do | + | - | - | + | + | + | - | - |
| Chip 1 | 5.4 | + | - | - | + | + | + | - | - |
| Chip 2 | 5.6 | + | - | - | + | + | + | - | - |
| Chip 3 | 5.9 | + | - | - | + | + | + | - | - |
| Chip 4 | 6.1 | + | - | - | + | + | + | - | - |
Note: "+" positive result in the table, "-" negative result.Sheet base reflectivity (%) is detected by Chengdu metrological supervision verification test.
Embodiment 2: fluorescence shows body detection method and black " coarse " biochip
In the present embodiment, used biochip is for containing black " coarse " biochip of black " coarse " sheet base, and used colorant, the label that contains colorant are identical with embodiment 1 with ginseng photo-based 0.
The present embodiment step is as follows:
Prepared black " coarse " the sheet base of present embodiment is coating-structure sheet sheet base.Coating is formed by pitch-dark, and the structure sheet is the transparent microslide (U.S. Erie Scientific company) of size 75 * 25 * 1mm.Present embodiment used pitch-dark be automobile finish (Peal blackberry, Qifu Industrial Development Co., Ltd., Shanghai).Spray pitch-darkly at the microslide end face, dry back forms black coating (thickness is about 30um), and note is made sheet base 21.The roughness of sheet base 21 is (being detected by Chengdu metrological supervision verification test).
The preparation of black " coarse " biochip, except that black " coarse " the sheet base that uses the present embodiment preparation, the preparation method is identical with preparation method in the example 1.
Present embodiment fluorescence shows the body detection method, and except that black " coarse " the sheet base that uses the present embodiment preparation, other is all identical with example 1 as fluorescent marker, sample, detection step.
With example 1, scanner is confocal laser scanner (GMS of Afymetrix company 418 chip scanners), scanning excitation wavelength 532nm, and wavelength of transmitted light 570hm, the treated software of the signal that reads (AfymetrixJAGUAR 2.0) is handled.Gained result such as table 2.
Table 2 fluorescence shows the testing result of body biochip
| Chip | Sheet base roughness | HCV antibody positive serum | HIV antibody positive serum | Positive control | The negative control thing | ||||
| HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | ||
| Chip 0 | Do not do | + | - | - | + | + | + | - | - |
| Chip 21 | 0.47um | + | - | - | + | + | + | - | - |
Note: "+" positive result in the table, "-" negative result.Sheet base roughness is detected by Chengdu metrological supervision verification test.
Embodiment 3: complex light shows body detection method and white biochip
In the present embodiment, prepared and used biochip is the white biochip that contains the white tablets base, and used ginseng photo-based 0 is identical with embodiment 1.
The present embodiment step is as follows:
The preparation of-white tablets base:
The prepared white background sheet base of present embodiment is: monobasic white material sheet base, white coating-structure sheet sheet base and transparent-white material composite sheet base.
The prepared monobasic white material sheet base of present embodiment is a white plastic sheet base, and it is the thermoplastic polyvinyl chloride after adding filling agent such as titanium dioxide, makes through compression molding, is of a size of 75 * 25 * 1mm, and below note is made sheet base 31.
White coating-structure sheet sheet base that present embodiment is prepared, coating is formed by whitewash, and the structure sheet is the transparent microslide (U.S. Erie Scientific company) of size 75 * 25 * 1mm.The used white paint of present embodiment is ocean, a river auto spray painting (Chengdu ruddiness Paint Factory).Spray-coated white paint on transparent microslide end face, dry back forms white coating.Formed tunica albuginea layer thickness is about 30um.More than the white tablets base of preparation note is made sheet base 32.
Transparent-white material composite sheet base that present embodiment is prepared, its transparent material is respectively the polyvinyl chloride film of 75mm * 25mm * 15um (long * wide * thick) and with reference to substrate 0, its white material is respectively white plastic sheet base 31 and white coating (the automatic white of Chuan Yang is sprayed paint, Chengdu ruddiness Paint Factory).The polyvinyl chloride film thermoplastic is fitted in the end face of white plastic sheet base, and the sheet base note that makes is made sheet base 33.The white auto spray painting is sprayed on the bottom surface of transparent microslide, is dried to white films, and the sheet base note that makes is made sheet base 34.
The preparation of-white biochip:
The preparation of the white biochip of present embodiment, except that the white tablets base that uses the present embodiment preparation, the preparation method is identical with preparation method in the example 1.Reactor wherein, be to be formed on the white plastic sheet base end face on 31, being to be formed on the white coating surface, sheet base top on the sheet base 32, is being to be formed on the sheet base top transparent film surface on the sheet base 33, is being to be formed on the sheet base top amination surface of glass slide on the sheet base 34.
In this example, used probe is HCV antigen (the People's Hospital, BeiJing, China hepatopathy research institute) and HIV1+2 antigen (the People's Hospital, BeiJing, China hepatopathy research institute).In the reactor of each above-mentioned preparation, respectively HCV antigen (1.5mg/ml) and HIV1+2 antigenic solution (1.5mg/ml) point sample are fixed.In a reaction tank, 2 diameters of two kinds of antigen each points are the point of 200um, form 2 * 2 arrays.After bag is done, standby with the confining liquid sealing.Prepared chip, according to employed base numbering, note is done chip 31, chip 32, chip 33, chip 34 respectively.
-complex light shows the body detection method:
In the present embodiment, the goat-anti people two anti-(Fujian Changli Biochem. Co., Ltd., Quanzhou City) that used coloured label is a colloid gold label, used signal amplification agent is silver-colored reinforcing agent (a Sigma company), specimen in use is with embodiment 1.
4 kinds of samples add respectively in the reactor of said chip during experiment.In 4 reactors of each biochip, be added with the sample of dilution in 1: 50 respectively.The application of sample amount is 5ul.React after 5 minutes and wash 5 times, the each addition of cleansing solution is 15ul.
Coloured label addition is 5ul, reaction back washing 5 times, and the each addition of cleansing solution is 15ul, dry laggard line scanning.Scanner is EPSON 1260 scanners (EPSON companies), and the scanning irradiation light is a white light, and signal light is white light also, and the treated software of the signal that reads (Afymetrix JAGUAR 2.0) is handled, and obtains reaction result (table 3) after averaging then:
Table 3, white light shows the testing result of body detection chip
| Chip | Sheet base reflectivity (%) | HCV resists this positive serum | HIV antibody positive serum | Positive control | The negative control thing | ||||
| HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | ||
| Chip 0 | Do not do | + | - | - | + | + | + | - | - |
| Chip 31 | 61 | + | - | - | + | + | + | - | - |
| Chip 32 | 57 | + | - | - | + | + | + | - | - |
| Chip 33 | 55 | + | - | - | + | + | + | - | - |
| Chip 34 | 56 | + | - | - | + | + | + | - | - |
Note: "+" positive result in the table, "-" negative result.
Embodiment 4: the biochip test method of applying nano colored materials biochip and nanometer colored materials biochip kit
In the present embodiment, prepared and used biochip is the white biochip that contains the white tablets base, and used ginseng photo-based 0 is identical with embodiment 1.
The present embodiment step is as follows:
The preparation of-nanometer colored materials sheet base:
Prepared sheet base is a nanometer white material sheet base in the present embodiment, comprises sheet base and nano particle the sheet base in sheet base whole nonferrous layer of nano particle on sheet base end face top layer.
The prepared nano particle of present embodiment is at the sheet base on sheet base end face top layer, employed white substrate is the white coating sheet base (sheet base 32) of preparation among the embodiment 3, employed nano material is the silicon oxide nanoparticle liquid (mean grain size 40nm, Sigma-Aldrich company) of own high degree of dispersion.The sheet base 41 that present embodiment is prepared, be will be in advance the silicon oxide nanoparticle liquid of dilution be coated on the white organic coating layer surface of substrate 32 and make.
The sheet base 42 that present embodiment is prepared, be will be in advance the silicon oxide nanoparticle liquid of dilution be coated on the white organic coating layer surface of substrate 32 and make in the regional area.Coating place of silicon oxide nanoparticle liquid forms sheet Ji Chi, and isolation structure then is the hydrophobic relatively white paint coating surface that the end applies.Per 1 sheet base forms 8 sheet Ji Chi, and each sheet base pool size is 4.5mm * 4.5mm, and sheet base cell compartment is 4.5mm from structure width.
The prepared nano particle of present embodiment is fixed on the sheet base in the whole white layer of sheet base, be silicon oxide nanoparticle liquid (mean grain size 40nm with own high degree of dispersion, Sigma-Aldchi company), with selected ratio with after the foreign auto spray painting in river (Chengdu ruddiness Paint Factory) mixes, be coated in the end face of transparent microslide (U.S. ErieScientific company), drying makes again.Prepared base note made sheet base 43.
The preparation of-nanometer colored materials biochip:
Substrate 42 in the present embodiment is with through having isolation structure.Substrate 41 and 43 in the present embodiment as the sheet base in the example 1, leads on sheet base end face with high hydrophobic organosilicon coating (Chengdu morning twilight chemical design institute) formation reactor isolation structure.Form 8 ponds on each sheet base, each pond is of a size of 4.5mm * 4.5mm, and cell compartment is 4.5mm from structure width.
In this example, used probe is HCV antigen (the People's Hospital, BeiJing, China hepatopathy research institute) and HIV1+2 antigen (the People's Hospital, BeiJing, China hepatopathy research institute) with embodiment 1.In the reactor of each above-mentioned preparation, respectively HCV antigen (1.5mg/ml) and HIV1+2 antigenic solution (1.5mg/ml) point sample are fixed.In a reaction tank, 2 diameters of two kinds of antigen each points are the point of 200um, form 2 * 2 arrays.After bag is done, standby with the confining liquid sealing.Prepared chip, according to employed base numbering, note is done chip 41, chip 42 and chip 43 respectively.
-complex light shows the body detection method:
In the present embodiment, used label and sample are with embodiment 3.In the present embodiment, sample suitably dilutes, and other detection method is with embodiment 3.
In the present embodiment, scanner is EPSON 1260 scanners (EPSON companies), and the scanning irradiation light is a white light, and signal light also is white light, the treated software of the signal that reads (Afymetrix JAGUAR 2.0) is handled, and obtains reaction result (table 4) after averaging then:
The testing result of table 4 nanometer white material biochip
| Chip | Sheet base reflectivity (%) | HCV antibody positive serum | HIV antibody positive serum | Positive control | The negative control thing | ||||
| HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | ||
| Chip 0 | Do not do | + | - | - | + | + | + | - | - |
| Chip 41 | 56 | + | - | - | + | + | + | - | - |
| Chip 42 | 56 | + | - | - | + | + | + | - | - |
| Chip 43 | 56 | + | - | - | + | + | + | - | - |
Note: "+" positive result in the table, "-" negative result.
Embodiment 5: the biochip test method of applying nano particulate colored marker and contain the biochip kit of nanoparticle colored marker
In the present embodiment, the used ginseng photo-based 0 of institute is identical with embodiment 1, and preparation and used biochip are nanometer white material biochip 41.
The present embodiment step is as follows:
The preparation of-nanoparticle colored marker:
In the present embodiment nanoparticle colored marker, the mark aglucon is goat-anti people two anti-(Tiantan Bio-pharmaceuticals goods incorporated company), colorant is crystal violet (Long Huagongshijichang of Chengdu section), and nanoparticle is that mean grain size is the collaurum (Fujian Changli Biochem. Co., Ltd., Quanzhou City) of 35nm.
The preparation of present embodiment coloured marking particulate is that colorant, mark aglucon are mixed according to predetermined concentration and ratio with nanoparticle, at room temperature reacts 18 hours.Then, under 5000r/min centrifugal 5 minutes, remove supernatant.With the PBS washing, centrifugal again, three times repeatedly, standby in PBS at last again.
-complex light shows the body detection method:
The biochip that uses in the present embodiment is the nanometer white material biochip 41 that is fixed with HCV antigen and HIV1+2 antigen probe, and the nanoparticle colored marker that is added is the crystal violet nanoparticle.
In the present embodiment, specimen in use and detection method are same as embodiment 3.
In the present embodiment, scanner is EPSON 1260 scanners (EPSON companies), and the scanning irradiation light is a white light, and signal light also is white light, the treated software of the signal that reads (Afymetrix JAGUAR 2.0) is handled, and obtains reaction result (table 5) after averaging then.
Table 5, the testing result of nanoparticle colored marker biochip kit
| Chip | Sheet base reflectivity (%) | HCV antibody positive serum | HIV antibody positive serum | Positive control | The negative control thing | ||||
| HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | HCV antibody | HIV antibody | ||
| Chip 0 | Do not do | + | - | - | + | + | + | - | - |
| Chip 41 | 56 | + | - | - | + | + | + | - | - |
Note: "+" positive result in the table, "-" negative result.
Claims (21)
1, a kind of method that object in the sample is carried out qualitative and/or quantitative test, it comprises following basic step:
A, object or the material that contains object are contacted with the reactor of pigmented biological chip and reaction therein, described have the color chip reactor to contain the colored pellets of coloured background base is provided, and the density of the probe of colored pellets base or probe-particulate is greater than 2 point/square centimeters in the described reactor;
B, colorant or the material that contains colorant are anchored at reaction place in the described reactor of a, to be formed with Semu mark;
C, the monochromatic or compound irradiation light of wavelength between 200-780nm is applied in the described reactor of b;
D, to light, coloured background and coloured target in the described reactor of c and between or the signal light that produces of interaction between the signal detection device detect;
E, according to the described described reaction result of interpretation of result a that detects of d;
Wherein: in described interaction, form color code between described coloured background and the coloured target or/and saturation degree or/and the contrast of brightness.
2, a kind of method of the object in the sample being carried out qualitative and/or quantitative test according to claim 1, it is characterized in that: when the main signal light that provides by colorant on described coloured target, described coloured background of base to the light reflectivity of signal light less than 10%.
3, a kind of method of the object in the sample being carried out qualitative and/or quantitative test according to claim 2, it is characterized in that: described colorant is for providing the material of the signal light on described coloured target, and described base is that its coloured background is dark sheet base under white light.
4, a kind of method of the object in the sample being carried out qualitative and/or quantitative test according to claim 1, it is characterized in that: when mainly providing signal light on described coloured target by the selective reflecting of colorant, described coloured background of base to the selective reflecting rate of signal light less than 10% or its non-selective reflectivity greater than 50%.
5, a kind of method that object in the sample is carried out qualitative and/or quantitative test according to claim 4 is characterized in that: described colorant is monochromatic or approaching monochromatic colorant; Described base is that its coloured background is light color under white light.
6, a kind of method of the object in the sample being carried out qualitative and/or quantitative test according to claim 1, it is characterized in that: when the non-selective reflection that mainly contains colorant provided signal light on described coloured target, the non-selective reflectivity of the coloured background of described substrate was greater than 50% or less than 10%.
7, a kind of method that object in the sample is carried out qualitative and/or quantitative test according to claim 6 is characterized in that: affiliated colorant is black or dark colorant, and described base is that its coloured background is light color under white light; Perhaps described colorant is light color or white colorant, and described base is that its coloured background is dark sheet base under white light.
8, a kind of biochip, it is characterized in that: include color chips base and probe in its reactor at least or/and the probe particulate, the probe in the described reactor on the colored pellets base or the density of probe-particulate are greater than 2 point/square centimeters, wherein: in described interaction, form color code between described coloured background and the coloured target or/and saturation degree or/and the contrast of brightness.
9, a kind of biochip according to claim 8 is characterized in that: described colored pellets base comprises monobasic colored materials sheet base, colored coating-structure sheet sheet base, coloured diaphragm-structure sheet sheet base, minimum reflected structural sheet-coloured structure sheet sheet base, sees through-colored materials sheet base and nanometer colored materials sheet base.
10, a kind of biochip according to claim 9, it is characterized in that: form in colored materials, transparent material and the filter of described coloured background in its sheet base, contain following one or more organism: nitrocellulose, polystyrene, polyacrylate, polysulfones, polyethersulfone, Polyvinylchloride, amino resins.
11, a kind of biochip according to claim 9 is characterized in that: in the described nanometer colored materials sheet base, described nanoparticle mean grain size is 1-500nm, be distributed in sheet base part or all surfaces or be distributed in the colored materials layer.
12, a kind of biochip according to claim 11, it is characterized in that: described nanoparticle, comprise gold, vanadium, lead, silver, iron and oxide fine particle thereof, monox, titanium dioxide, alumina particulate, plastics, polysaccharide, latex, resin comprise that also above-mentioned particulate modification is combined with the particulate derivant of functional group and/or function thing.
13, a kind of biochip according to claim 8 is characterized in that: described probe comprises that part, aglucon, antigen, antibody, polypeptide, protein and strand or multichain DNA, RNA, nucleotide, described probe-particulate are that mean grain size is of a size of the particulate of 0.1nm-10um and the connector of probe.
14, according to Claim 8 described a kind of biochip one of-13, it is characterized in that: stationary probe on its colored pellets base is or/and probe particulate place and peripheral region thereof, under white light, look it is following a kind of color: black, predominant wavelength is the dark color of redness, green, blueness, cyan or purple, white, and predominant wavelength is the light color of redness, yellow, green, blueness, cyan or purple.
15, a kind of biochip according to claim 8 is characterized in that: the roughness Ra on surface that coloured background is provided on the described colored pellets base is between 0.02-3.0um.
16, a kind of biochip kit is characterized in that: the material that it contains the biochip and the colorant of colored pellets base or contains colorant; Wherein: in described interaction, form color code between described coloured background and the coloured target or/and saturation degree or/and the contrast of brightness.
17, a kind of biochip kit according to claim 16, it is characterized in that: described colorant comprises fluorescent material, chemiluminescence catalyzer, colour developing and luminous substrate, non-ferrous metal or non-ferrous metal salt, dyestuff and pigment, and the described material that contains colorant comprises colored marker, nanoparticle colored marker and molecular vehicle colored marker.
18, a kind of biochip kit according to claim 17 is characterized in that: its colorant is one of the following kind or multiple material: fluorescein, rhodamine, amino black, Coomassie brilliant blue, silver salt, crystal violet, colloid or nm of gold, electroselenium, modified carbon black, titanium dioxide, chemiluminescence catalyzer, water soluble dyestuffs, water oil zwitterionic dyestuff, color paste of printing coating.
19, a kind of biochip kit according to claim 17 is characterized in that: described nanoparticle colored marker, nanoparticle mean grain size wherein is 1-500nm.
20, a kind of biochip kit according to claim 19, it is characterized in that: described nanoparticle, comprise gold, vanadium, lead, silver, iron and oxide fine particle thereof, monox, titanium dioxide, alumina particulate, plastics, polysaccharide, latex, resin comprise that also above-mentioned particulate modification is combined with the particulate derivant of functional group and/or function thing.
21, a kind of biochip kit according to claim 20, it is characterized in that: described functional group comprises amino, acyl group, carboxyl, hydroxyl, aldehyde radical and epoxy radicals.
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| CNB031176453A CN100347545C (en) | 2003-04-08 | 2003-04-08 | Method of preoceeding qualitative and/or quantitative analysis against target substance in sample and its detecting device |
| PCT/CN2003/001091 WO2004081570A1 (en) | 2003-03-13 | 2003-12-19 | A chip matrix, a chip comprising the matrix and their preparation and application |
| AU2003289617A AU2003289617A1 (en) | 2003-03-13 | 2003-12-19 | A chip matrix, a chip comprising the matrix and their preparation and application |
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| CN1735807A (en) * | 2003-12-19 | 2006-02-15 | 成都夸常医学工业有限公司 | Chip detecting method and relevant apparatus |
| CN1648671B (en) * | 2005-02-06 | 2012-09-26 | 成都夸常医学工业有限公司 | Detecting method for multiple reactor analytic chip and analytic chip and detector |
| CN1297819C (en) * | 2004-09-29 | 2007-01-31 | 南京大渊生物技术工程有限责任公司 | Biological chip quantitative detecting method |
| GB2464747B (en) | 2008-10-10 | 2013-05-15 | Hai Kang Life Corp Ltd | Method for detection of analyte in microarray of samples and apparatus for performing such method |
| CN101865926B (en) * | 2010-06-13 | 2013-09-11 | 云南中医学院 | Detection method for trace protein of Chinese medicinal injection |
| CN103018439B (en) * | 2012-11-30 | 2013-12-25 | 中国农业科学院油料作物研究所 | Method for enhancing detection sensitivity of colloidal gold immunity chromatography test strip |
| CN103197055B (en) * | 2013-03-12 | 2015-04-15 | 王颖颖 | Method for reducing background of vector in biological reaction |
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| US6025202A (en) * | 1995-02-09 | 2000-02-15 | The Penn State Research Foundation | Self-assembled metal colloid monolayers and detection methods therewith |
| US20020128234A1 (en) * | 1999-04-28 | 2002-09-12 | Hubbell Jeffrey A. | Multifunctional polymeric surface coatings in analytic and sensor devices |
| EP1296139A1 (en) * | 2000-06-20 | 2003-03-26 | Mitsubishi Rayon Co., Ltd. | Micro-array of organism-associated substance |
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| US6025202A (en) * | 1995-02-09 | 2000-02-15 | The Penn State Research Foundation | Self-assembled metal colloid monolayers and detection methods therewith |
| US20020128234A1 (en) * | 1999-04-28 | 2002-09-12 | Hubbell Jeffrey A. | Multifunctional polymeric surface coatings in analytic and sensor devices |
| EP1296139A1 (en) * | 2000-06-20 | 2003-03-26 | Mitsubishi Rayon Co., Ltd. | Micro-array of organism-associated substance |
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