CN102890101B - Biosensor chip having self-calibration regions and testing method of same - Google Patents
Biosensor chip having self-calibration regions and testing method of same Download PDFInfo
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- CN102890101B CN102890101B CN201110200327.XA CN201110200327A CN102890101B CN 102890101 B CN102890101 B CN 102890101B CN 201110200327 A CN201110200327 A CN 201110200327A CN 102890101 B CN102890101 B CN 102890101B
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Abstract
The invention relates to a biosensor chip having self-calibration regions and a testing method of the same. The biosensor chip comprises a testing region and at least one calibration region, wherein the testing surface of the testing region is modified with capture molecules for capturing to-be-tested molecules, the testing surface of each calibration region is modified with a predetermined number of calibration molecules and corresponding reference values, the testing region has a testing output for outputting testing signals of the testing region, and each calibration region has a calibration output for outputting testing signals of the calibration region. According to the invention, the biosensor chip simultaneously comprises the testing region and the calibration regions, and thus can realize self-calibration of the sensor chip.
Description
Technical field
The present invention relates to the biologic sensor chip for chemistry and the detection of two packing spaces product.More specifically, the present invention relates to a kind of biologic sensor chip and calibration steps thereof with self-calibration function.
Background technology
Owing to using the development of the micro-fluidic technologies of micro solution, make the purposes of lab-on-a-chip (LOC) or micro-fluid chip be departed from the constraint of traditional research experiment, further march toward compared with the business application of tool practical value.In the field such as chemical, biological, comprise that the microfluid system of micro-fluid chip is more and more applied, as synthetic in the transmission of micro fluid, minor compound, sample separation, constituent analysis and chemical reaction.
By biologic sensor chip is loaded in microfluid sample boat, make the test surfaces of biologic sensor chip as a surface of example boat reaction chamber, can realize the measurement to test agent.In utilization, utilize in the measuring system that electromagnetic biological sensor chip measures, even target molecular conecentration is constant in test agent, the test result of electromagnetic biological sensor also can there is a strong possibility along with the difference of sensor chip and the difference of test environment and change.Cause that the factor that test result changes comprises: thick variation, the variation of chip lateral dimension and the variation of magnetic characteristic of each tunic of chip being caused by the drift of chip manufacturing process; The variation of temperature of reaction during test, the variation of reagent flow velocity in example boat microchannel, the variation of reagent volume, reagent quality is as the variation of magnetic granular mass, the caused change in flow of variation of the size of microfluidic device critical size such as microchannel and the size of reaction chamber, reagent volume variation etc.These factors can cause the inconsistency of test result.
In the chip manufacturing proces of for example magnetosensitive biologic sensor chip, the variation of the variation that each tunic of the chip that the drift of fabrication process parameters can cause is thick, the variation of lateral dimension and magnetic characteristic.When the layers of material thickness of magnetosensitive biologic sensor chip changes, the distance between magnetic particle and magneto-dependent sensor will change thereupon.Magnetic particle from the distance of sensor more away from, the probability being detected is less.Otherwise, magnetic particle from the distance of sensor more close to, the magnetic amounts of particles being detected is more.The variation of transducer transversely size also can cause the variation of magneto-dependent sensor detectability, and this is because the sensitivity of magnetosensitive device is relevant with size, shows as a kind of very complicated relation.In general, when other parameter constants, the detectability of sub-micron magneto-dependent sensor chip reduces along with reducing of size.The drift of fabrication process parameters more can make magnetic characteristic such as coercive force, saturation magnetic field intensity and the magnetic hysteresis loop etc. of magneto-dependent sensor chip change.When coercive force becomes large, detection sensitivity reduces.Equally, when saturation magnetic field intensity increases, detection sensitivity also can reduce.Another parameter is the drift of device magnetic hysteresis loop mid point.As magnetosensitive biology sensor, magnetic hysteresis loop midrange is the smaller the better from the distance at zero point, is preferably zero Gauss.If it is too far away to depart from zero point, transducer sensitivity can reduce, even can lose detectability.
The temperature of reaction of testing sample and reaction reagent can exert an influence to test result equally.Within being applicable to the temperature range of biochemical reaction, reaction efficiency corresponding rising along with the rising of temperature in general.If magnetosensitive biology sensor is at room temperature to use, have different room temperatures in country variant, different regions, varying environment, different seasons.For example, when temperature of reaction drops to 20 degrees Celsius from 30 degrees Celsius, testing sample and various reaction reagents enter the one-step reaction efficiency that reaction compartment reacts simultaneously can reduce by 50% left and right, and testing sample and various reaction reagent enter respectively reaction efficiency in the situation of three step successive reactions of reaction compartment can be reduced to 12.5%.
In the micro fluid reaction system of for example microfluid sample boat, the variation of reagent flow velocity, the variation of reagent volume also can affect reaction result.When in general, reagent flow velocity increases, reaction efficiency can reduce.The reaction molecular total amount that participates in reaction when reaction reagent volume increases increases, and shows as the increase of reaction efficiency.If wash reagent, the increase of irrigation flow rate and flush volume can be promoted developing result, affects reaction result.
In prior art, disclose the multiple biochip calibration way based on Measurement for Biotechnique, but these methods all can not be for the calibration of magnetosensitive biologic sensor chip.
The people's such as Grenville Arthur Robinson denomination of invention is " Capiliary-Fill Biosensor Device Compresing a Calibration Zone ", the US Patent No. 6 that on February 22nd, 2000 is announced, 027,944 disclose a kind of biosensor arrangement that comprises calibration areas, as shown in Figure 1.In Fig. 1, the 12Ji region, region 9 in T district is for test function designs, the reagent pad that wherein region 12 is used for bearing test reagent, and region 9 is for carrying reaction base wad.Testing sample enters into after cavity 6, reagent on region 12 is dissolved and be deposited on region 9, with target molecules and the reaction of the capture molecules on region 9 in testing sample, the signal of the signal intensity of target molecular conecentration in region 9 places generation has sign testing sample.10He region, region 14 in Zone R forms calibration region.The test agent that enters cavity 6 simultaneously on region 14, carry for the agent dissolves of calibrating to region 10.Signal and target molecules concentration that 10 places, region produce are irrelevant, are just designed to produce calibrating signal.Equally, in S district, 11He region, region 16 is another group calibration region.In this patent, although there is no physical isolation wall between T district, Zone R, S district, but because the height reagent very little and on region 12,14 and 16 of cavity 6 has not had horizontal liquid flow while starting to dissolve, the process that there is no reagent mix between each district, cavity 6 can be divided into independently test section, calibration areas, additional calibration district.But in adopting the fluidic structures of multistep chemical reaction, various reaction reagents are mixed by intentional or unintentional conventionally, are difficult to meet the requirement that different types of reagent is present in zones of different.So measure in structure at the microfluid that adopts multistep chemical reaction, the technical scheme of this employing subregion calibration can not provide the accurate calibration to microfluid sample boat and test chip.
The people's such as Mark Thompson name is called disclosed U.S. Patent application US20100204062 of " Calibration methods for multiplexed sensor arrays ", on August 12nd, 2010, disclose a kind of for calibrating the method for nano-sensor reactant response, as shown in Figure 2.There is shown nanometer line sensor, above nanowire sensor is NW fixed member, is modified with the material of catching testing molecule on NW fixed member, is used in addition catching the material of calibration molecule on nanometer fixed member simultaneously.If the concentration of known calibration molecule, the signal that calibration molecule produces can be used to the various factors of calibrating nanowire sensor and may exerting an influence to the accuracy of sensor.But concerning magneto-dependent sensor, magnetosensitive device can not be distinguished the signal of the magnetic particle marker deposits yields of calibration on molecule and the signal that the magnetic particle on testing molecule produces, so this technical scheme is not suitable for and take the magneto-dependent sensor that magnetic particle is label.
The people's such as Cary James Miller denomination of invention is " Immunoassay Device with Immuno-reference Electrode ", the US Patent No. 7723099 that on May 25th, 2010 is announced, discloses a kind of immunosensor system of using electrochemical phenomena to carry out Molecular Detection.This immunosensor system has two sensor chips, and one is the test chip for sampling test signal, and another is for the calibration chip of the background noise being caused by non-specific absorption is provided.By the test signal producing on test chip, deduct background signal on calibration chip and only obtain and the concentration dependent information of testing molecule, reduced disturbing effect and the impact of some other effect on test result in immunoassays, see Fig. 3.But this test macro need to have two chips of the test of being respectively used to and calibration.With respect to single chip architecture, cost increases, and produces degree of difficulty and strengthens, and need two chips itself to have higher consistance.
Therefore, need a kind of biologic sensor chip simple in structure with self-calibration function, particularly magnetosensitive biologic sensor chip.
Summary of the invention
In order to solve the problems of the technologies described above, realize the self calibration of sensor chip, according to the test surfaces of biologic sensor chip of the present invention, be divided into test zone and at least one calibration region.The test surfaces of calibration region is modified with the calibration molecule of predetermined quantity.According to the attribute of testing molecule, and binding ability testing molecule and seizure molecule, testing molecule and detection molecules, can select to be modified at the type of the calibration molecule on the test surfaces of calibration region, to calibrate affecting the factor of test result.The calibration molecule being modified in calibration region can be testing molecule, detection molecules, analog capture molecule or simulation testing molecule.Because it is known to be modified at the quantity of the calibration molecule in calibration region, the normal output valve that the calibration molecule of this predetermined quantity produces through test reaction can calculate according to gross data or empirical data, as the reference value of this calibration region.During test, measure respectively the output signal of test zone as test signal, measure the output signal of each calibration region as calibrating signal.Calibrating signal based on calibration region and the reference value of this calibration region, can determine the calibration factor of this calibration region.With each calibration factor obtaining, test signal is calibrated, can obtain measurement result accurately.
According to an aspect of the present invention, provide a kind of biologic sensor chip, this biologic sensor chip comprises test zone and at least one calibration region,
The test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule,
The test surfaces of each calibration region is modified with the calibration molecule of predetermined quantity and has corresponding reference value,
Described test zone has the test output of the measuring-signal of output test zone,
Each calibration region has the calibration output of the measuring-signal of this calibration region of output.
Preferably, described test zone and described at least one calibration region have identical size transverse to fluid flow direction, locate described test zone and described calibration region and make when this biologic sensor chip is placed in the reaction chamber of microfluid sample boat, the fluid flow state on regional test surfaces is identical.
Preferably, described at least one calibration region comprises that its test surfaces is modified with the calibration region of the detection molecules for detection of testing molecule of predetermined quantity, its test surfaces and is modified with the calibration region of the testing molecule of predetermined quantity, the calibration region of analog capture molecule that its test surfaces is modified with predetermined quantity and at least one in the calibration region of the simulation testing molecule that its test surfaces is modified with predetermined quantity.
Preferably, described at least one calibration region comprises that its test surfaces is modified with the calibration region of the testing molecule of predetermined quantity, this biologic sensor chip has correction factor, and this correction factor is determined by the ratio of the binding ability of described seizure molecule and described testing molecule and the binding ability of described testing molecule and described detection molecules.
Preferably, described at least one calibration region comprises that its test surfaces is modified with the calibration region of the simulation testing molecule of predetermined quantity, this biologic sensor chip has correction factor, and this correction factor is determined by analog capture molecule and the ratio of the binding ability of simulation testing molecule and the binding ability of simulation testing molecule and analog detection molecule.
Preferably, described biologic sensor chip is with the serve as a mark magnetosensitive biologic sensor chip of thing of magnetic particle.
Preferably, the calibration molecule in described at least one calibration region has uniform surface density.
According to a second aspect of the invention, provide a kind of microfluid sample boat, comprise biologic sensor chip as mentioned above.
According to a third aspect of the invention we, a kind of method of testing of biologic sensor chip is provided, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the calibration molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
Make the testing sample that contains testing molecule enter reaction compartment,
The detection reagent that makes to contain for detection of the detection molecules of testing molecule enters reaction compartment,
Make label enter reaction compartment,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
According to a forth aspect of the invention, a kind of method of testing of biologic sensor chip is provided, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the calibration molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
Make the testing sample that contains testing molecule, the detection reagent and the label that contain for detection of the detection molecules of testing molecule enter reaction compartment simultaneously;
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
Preferably, the calibration molecule in described calibration region is described molecules detected or described detection molecules.
Preferably, the calibration molecule in described calibration region is testing molecule, and this biologic sensor chip has the definite correction factor of ratio by the binding ability of the binding ability of described seizure molecule and described testing molecule and described testing molecule and described detection molecules,
This method of testing further comprises the cleaning step before measuring process, and
With described correction factor to described calibration after test signal revise, obtain revising rear test signal.
According to a fifth aspect of the invention, a kind of method of testing of biologic sensor chip is provided, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the analog capture molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
The calibrating reagent of the simulation testing molecule that makes to contain concentration known and the testing sample that contains testing molecule enter reaction compartment,
The detection reagent that makes the analog detection reagent that contains analog detection molecule and contain for detection of the detection molecules of testing molecule enters reaction compartment,
Make label enter reaction compartment,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
According to a sixth aspect of the invention, a kind of method of testing of biologic sensor chip is provided, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the analog capture molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
The testing sample that makes to contain testing molecule, contain detection reagent for detection of the detection molecules of testing molecule, calibrating reagent, the analog detection reagent that contains analog detection molecule and the label of the simulation testing molecule that contains concentration known enter reaction compartment simultaneously
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
Preferably, described in, containing the calibrating reagent of simulating testing molecule is liquid reagent or powdered reagent.
According to a seventh aspect of the invention, a kind of method of testing of biologic sensor chip is provided, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the simulation testing molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
Make the testing sample that contains testing molecule enter reaction compartment,
The detection reagent that makes the analog detection reagent that contains analog detection molecule and contain for detection of the detection molecules of testing molecule enters reaction compartment,
Make label enter reaction compartment,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
According to an eighth aspect of the invention, a kind of method of testing of biologic sensor chip is provided, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the simulation testing molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
The testing sample that makes to contain testing molecule, contain the detection reagent for detection of the detection molecules of testing molecule, the analog detection reagent that contains analog detection molecule and label and enter reaction compartment simultaneously,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
Beneficial effect of the present invention is:
The present invention is divided into test zone and calibration region by biologic sensor chip, as required at the calibration molecule of calibration region finishing predetermined quantity, respectively test zone and calibration region are measured, the calibration factor being obtained by the normal reference value of the calibrating signal based on calibration region and calibration region is realized the calibration to test signal.According in sensor chip of the present invention, decorating molecule in test zone and each calibration region is in identical reaction environment and reaction conditions, resulting calibration factor has characterized reaction environment in biomolecule measurement, the impact that the structure of multi-step reaction conditions and chip structure and microfluid sample boat produces test signal exactly.With the calibration factor obtaining, test result is calibrated, can eliminate the manufacture craft of sensor chip, the impact that the various factorss such as specimen in use boat, reaction reagent, test environment and method of testing produce the test result of testing sample, obtains accurately, has good conforming test result.According to biologic sensor chip of the present invention, test zone and calibration region coexist in same reaction compartment, between the test test surfaces of zones of different, there is no physical isolation, have simple in structurely, and measurement result is accurate, consistance and reproducible feature.
Accompanying drawing explanation
Below with reference to accompanying drawings to advantage of the present invention, feature and beneficial effect are specifically described, wherein
Fig. 1 is the biosensor structure figure that prior art has calibration areas.
Fig. 2 is the calibrating principle schematic diagram of prior art nanowire sensor.
Fig. 3 is the calibration steps process flow diagram of prior art sensing system.
Fig. 4 is for the decomposing schematic representation of the microfluid sample boat of sensor chip principle of work is described.
Fig. 5 shows the schematic diagram of magnetosensitive biosensor test principle.
Fig. 6 is the fundamental diagram of the magnetosensitive biologic sensor chip of the example 1 according to the present invention.
Fig. 7 is the fundamental diagram of the magnetosensitive biologic sensor chip of the example 2 according to the present invention.
Fig. 8 is the fundamental diagram of the magnetosensitive biologic sensor chip of the example 3 according to the present invention.
Fig. 9 is the fundamental diagram of the magnetosensitive biologic sensor chip of the example 4 according to the present invention.
Figure 10 is the fundamental diagram of the magnetosensitive biologic sensor chip of the example 5 according to the present invention.
Embodiment
Below in conjunction with drawings and Examples, summary of the invention of the present invention is further described.In accompanying drawing, same or analogous feature represents with corresponding or similar Reference numeral.
Fig. 4 illustrates for the decomposing schematic representation of the microfluid sample boat of sensor chip principle of work is described.This microfluid sample boat comprises the first sealant 100, solution storage chamber layer 101, the second sealant 102 and the reaction chamber that the set gradually microchannel layers 104 of holding concurrently.Solution storage chamber layer 101 comprises a plurality of liquid storage cylinders that run through this layer thickness, and liquid storage cylinder preferably has the structure that is used to form pump chamber.By being separately positioned on the first sealant 100 of liquid storage cylinder top, the reaction chamber that is arranged on chamber layer 101 belows hold concurrently microchannel layers 104 and the second sealant 102 therebetween, described a plurality of liquid storage cylinder forms a plurality of fluid reservoir 1012-1016, for holding respectively testing sample and the reaction reagent for testing sample is reacted.Reaction chamber is held concurrently in a side of microchannel layers 104 and is formed with the microchannel 1048 that is formed at liquid storage cylinder below, is formed with the reaction chamber being communicated with microchannel 1048, and passes through the waste liquid pool 1011 that microchannel 1047 is communicated with reaction chamber on opposite side.The sample or the reagent that are stored in each fluid reservoir enter microchannel by losing the second sealant of sealing function, in reaction chamber, carry out biochemical reaction, by the microbe chip being arranged in reaction chamber, reaction result is measured, realized the test to sample.Reacted liquid flows into waste liquid pool through microchannel.Lower port place at fluid reservoir 1012 preferably arranges filtering membrane 103, for filtering the material of test agent interference detection results.
Reaction chamber is positioned at the hold concurrently lower surface of channel layer 104 of reaction chamber, and its two ends are connected with microchannel 1047,1048 respectively.
For preventing that reagent from leaking, reaction chamber bottom can seal example boat with other thin film or flat board.Fig. 4 specifically illustrated by printed circuit board (PCB) 105 as seal pad structure, in the position of corresponding reaction chamber, be welded with biologic sensor chip 106.By the biologic sensor chip being opposite in reaction chamber, measure, can realize the measurement to testing sample.
Take below and adopt that in the reaction chamber of the microfluid sample boat shown in Fig. 4, magnetosensitive biology sensor according to the present invention to be installed be example, to describing according to the test philosophy of magnetosensitive biology sensor with according to the structure of magnetosensitive biologic sensor chip of the present invention and principle of work.Should be appreciated that microfluid sample boat shown in Fig. 4 and the magnetosensitive biochip that is applied to microfluid sample boat are that schematically biologic sensor chip of the present invention is applicable to have the microfluid sample boat of other structures.
Take below label as magnetic particle be example, the test philosophy of magnetosensitive biologic sensor chip is described.Fig. 5 shows the schematic diagram of magnetosensitive biosensor test principle.The magnetosensitive biologic sensor chip 506 that is modified with seizure biomolecule 5001 on it is applied to the printed circuit board (PCB) 105 shown in Fig. 4 and is placed in reaction chamber.By the output signal of survey sensor chip, can obtain the quantity of testing molecule in testing sample.The microchannel 1048 that testing sample in Fig. 4 fluid reservoir 1012 is held concurrently in microchannel layers 104 by reaction chamber is injected into reaction chamber, chip 506 surfaces of flowing through.Testing molecule 5002 in testing sample is attached to and is caught in biomolecule 5001 by biochemical reaction.Then, the microchannel 1048 that the detection reagent that contains detection molecules 5003 in fluid reservoir 1013 is held concurrently in microchannel layers 104 by reaction chamber is injected into reaction chamber, chip 506 surfaces of flowing through.Detection molecules 5003 and testing molecule 5002 react and combine.In detection molecules 5003, carry for example the first binding molecule 5004 of biotin (biotion) molecule, it is in conjunction with rolling into a ball 5003-5004 also referred to as detection molecules.After completion of the reaction, if needed, the cleaning fluid being stored in fluid reservoir 1014 can be used to clean chip 506 surfaces, to wash unconjugated detection molecules for detection molecules 5003 and testing molecule 5002.After this, the label magnetic particle reagents 5006 that is stored in the second binding molecule 5005 with for example Streptavidin (streptavidin) molecule in fluid reservoir 1015 is injected into reaction chamber, on chip 506 surfaces, by the combination of the first binding molecule and the second binding molecule, label magnetic particle and detection molecules 5003 combinations, finally realize the combination of testing molecule 5002 and label magnetic particle 5006.After magnetic particle reagents has reacted, be stored at wash liquid stream in fluid reservoir 1016 through chip 506 surfaces, depositing to but not the magnetic particle being reacted on chip surface rinse well.Certainly, testing sample as above, detection reagent and label reagent also can enter reaction chamber simultaneously, and obtain strand as shown in the figure on test chip surface.
An additional magnetic field, magnetize the magnetic particle being combined in molecules detected in this magnetic field.Magneto-dependent sensor chip 506 detects the quantity of testing molecule by detecting the signal of magnetic particle generation.
From the above description to magneto-dependent sensor principle of work, the difference due to the manufacture craft of the magnetosensitive biologic sensor chip of different batches, may cause the surface of different chips different to the reaction bonded ability of same reaction thing.Batch difference of reaction reagent is different with transportation storage condition, can cause the difference of binding ability between used reagent.Due to the difference of the manufacture craft of the microfluid sample boat of different batches, may cause between example boat microfluidic structures and the assembling of sensor chip and example boat there are differences.The environment temperature of test is different from the efficiency of magnetic particle combination to causing testing molecule with the complicated and simple meeting of reactions steps in test process.These differences and difference all can exert an influence to the test result of sample.Below with reference to accompanying drawings and in conjunction with specific embodiments structure and the principle of work with the sensor chip of self-calibration function according to the present invention described.
According to a preferred embodiment of the invention, biologic sensor chip comprises test zone and at least one calibration region, and test zone and each calibration region are exported respectively test signal and calibrating signal.The test surfaces of test zone is modified with for catching the biometric capture molecule of testing molecule.The biochemical reaction carrying out on this test zone and test philosophy and same or similar above with reference to the test philosophy described in Fig. 5.The signal that the output of test zone answers externally-applied magnetic field to produce for exporting the magnetic granular sensation of test zone combination.The test surfaces of each calibration region is modified with respectively a kind of calibration molecule of predetermined quantity.Calibration molecule can be testing molecule, detection molecules, analog capture molecule or simulation testing molecule.The biochemical reaction carrying out in calibration region is relevant with the type of modified calibration molecule.The signal that the output of calibration region answers externally-applied magnetic field to produce for exporting the magnetic granular sensation of calibration region combination.The type of the calibration molecule in calibration region determines, and the quantity of calibration molecule is when known, the normal signal value that the quantity of the magnetic particle of combination in this calibration region and induction externally-applied magnetic field produce according to theory, calculate or empirical data known.This normal signal value can be used as reference value R, for test data is calibrated.
The method of testing of biologic sensor chip according to the preferred embodiment of the invention, comprises the following steps:
Make the testing sample of testing molecule enter reaction compartment,
The detection reagent that makes to contain for detection of the detection molecules of testing molecule enters reaction compartment,
Make label enter reaction compartment,
Measure respectively the output signal of test zone and the output signal of each calibration region, obtain test signal A and at least one calibrating signal C; And
By the corresponding reference value R of calibrating signal He this calibration region of calibration region, can determine the calibration factor α of this calibration region, α=R/C;
With calibration factor α 1 described in each, α 2..., calibrates described test signal A, obtains calibrating rear test signal T=(α 1 α 2...) A.
Example 1
Because the surface reaction ability of different magnetosensitive biology sensors is different, the difference of the different and transportation condition of storage of reagent lot, can be due to reactive variation between the second binding molecule of magnetic-particle and the first binding molecule of detection molecules, cause the reaction capacity of label magnetic particle to change, affect the sensitivity of sensor chip.
Fig. 6 illustrates the principle of work schematic diagram of the magnetosensitive biologic sensor chip 606 of the example 1 according to the present invention.Magnetosensitive biologic sensor chip 606 surfaces comprise first area 6100 and second area 6200.First area 6100 is test zone, and its finishing has the molecule 6001 of seizure.The test reaction occurring on this first area with to the method for testing of this first area and Fig. 5 and identical to the explanation of Fig. 5.Detection molecules 6003 is for detection of testing molecule 6002.Second area 6200 is calibration region, and finishing has the detection molecules 6003 for detection of testing molecule 6002 of predetermined quantity.Using the detection molecules of this predetermined quantity in theory or the output signal that in experience, the magnetic particle of combination produces as the reference value of this calibration region.In detection molecules 6003, be combined with the first binding molecule 6004.When testing sample is flowed through chip 606 surperficial, 6002 of testing molecules and be present in seizure molecule 6001 reactions on first area 6100, are combined in and catch on molecule 6001.Testing molecule discord second area 6200 reacts thereby is not combined on second area.Equally, when detection molecules 6003 is passed through the first area of sensor chip 605, detection molecules 6003 and the testing molecule 6002 being present on first area 6100 react and are combined on testing molecule.Subsequently, with magnetic particle 6006 reagent of the second binding molecule 6005, flow through the surface of chip 606.The second binding molecule 6005 on label magnetic particle 6006 both with first area 6100 in the detection molecules first binding molecule reaction of carrying, again with second area 6200 in the detection molecules of the modifying first binding molecule reaction of carrying.The reaction of the second binding molecule 6005 and the first binding molecule 6004 is high-affinity rapid reactions, and the combination speed between them and Density Distribution are not subject to the impact of reacting between testing molecule 6002 and detection molecules 6003.Accurately the detection molecules of controlled modification on the second area 6200 of chip 606 rolled into a ball the surface density of 6003-6004 and controlled detection molecules group and be uniformly distributed on second area, can accurately control the surface density of magnetic particle in second area, can make thus the surface density of the magnetic particle of different magnetosensitive biologic sensor chip second area combinations be consistent.
The reagent of the different batches of reagent and different transportation conditions of storage, and different biochip manufacturing process can cause reactive variation, the i.e. variation of detection molecules and label magnetic particle binding ability between the first binding molecule 6004 that the second binding molecule 6005 that magnetic particle carries and detection molecules carry.When testing with magnetosensitive biologic sensor chip 606, the first binding molecule in first area 6100 and second area 6200 is identical with the conjugation condition between the second binding molecule, and above-mentioned variation is present in first area and second area the impact of measuring-signal simultaneously.By respectively first area and second area being measured, obtain the test signal of first area and the calibrating signal of second area.Reference value based on this calibration region and the calibrating signal measuring can be determined the calibration factor of this calibration region.The calibration factor that utilization obtains is calibrated the test signal of test zone, can obtain test signal and test result accurately through calibration.
Embodiment according to the present invention, by by the measurement surface subregion of biochip, finishing in calibration region has predetermined quantity and equally distributed detection molecules, can calibrate the impact difference of the difference of the variation of detection molecules binding ability, chip structure and example boat being produced test result due to label magnetic particle, improve the measuring accuracy of magnetosensitive biologic sensor chip.
The calibration region of this example can be used separately, also can be used in combination with the calibration region of other examples.
Example 2
When another changing factor relevant with magnetic particle is magnetic particle reaction in the distribution of reaction surface zones of different.In the fluidic structures of conventional microchannel, the flow velocity of magnetic particle in the middle of microchannel is greater than the flow velocity in both sides, microchannel or bottom, cause that magnetic particle piles up in both sides, microchannel gradually, cause the surface density of magnetic particle in the middle of microchannel to be less than the surface density at edge, microchannel, make measurement result can not correctly characterize the quantity of testing molecule in testing sample.
Can think, similar with shape for area in sensor chip, in reaction compartment two identical regions of fluid flow state of living in, magnetic particle density is also just identical on the impact of such Liang Ge district test result.Impact test result being produced for calibrating the variation of magnetic particle-surface density, can be to comprise the first area that is used as test zone and the second area that is used as calibration region by the surface segmentation of sensor chip, the size and shape in two regions is same or similar, and on it, fluid flow state is identical.
Fig. 7 illustrates the fundamental diagram of the magnetosensitive biologic sensor chip of the example 2 according to the present invention.Magnetosensitive biologic sensor chip 706 comprises first area 7100 and second area 7200.The shape of first area and second area is similar, identical transverse to the size fluid flow state identical and their surf zones of fluid flow direction.Line 7047A and 7048B between two slight path 10s 47 and 1048 corresponding border in figure, have been schematically shown in the reaction chamber of microfluid sample boat shown in Fig. 4.The fluid state along fluid flow direction that is appreciated that region between the outlet of microchannel 1048 in reaction chamber and the entrance of microchannel 1047 is identical.Can find out, near the accumulation of magnetic particle region 7047A and 7047B is equal to the impact on calibration region to the impact of test zone.
For example, when this example 2 and example 1 in conjunction with time, by respectively first area and second area being measured, obtain the test signal of first area and the calibrating signal of second area.Reference value based on this calibration region and the calibrating signal measuring can be determined the calibration factor of this calibration region.The calibration factor that utilization obtains is calibrated the test signal of test zone, can obtain calibrating the accurately test result of the difference of sensor chip, reaction reagent and example boat on the impact of test result.
It will be appreciated by those skilled in the art that in this example, the position relationship of microchannel and magnetosensitive biologic sensor chip is schematic but not determinate.By test zone and calibration region location being made when the reaction compartment of chip in microfluid sample boat, the fluid flow state of each area surfaces is identical, can calibrate impact test signal being produced due to the accumulation of magnetic particle in microchannel, further improve the measuring accuracy of sensor chip.
The test zone of this example and the location of calibration region can be combined with the technical scheme of other examples, to obtain technique effect of the present invention.
Example 3
As shown in Figure 5, when measurement when surface of magnetic grain flow through sensor chip 506, a part of magnetic particle is attached in detection molecules, a part of magnetic particle because gravity sedimentation to the surface of chip.Magnetic particle for fear of deposition affects test generation, need to wash the magnetic particle that deposits to chip surface with cleaning fluid.In cleaning process, flow velocity and the volume of cleaning fluid have a significant impact cleaning performance.Greatly cleaning performance is good for fast, the used cleaning fluid volume of flushing speed.The little effect that does not likely have cleaning of slow, the used cleaning fluid volume of cleaning speed.On the other hand, cleaning process also likely rinses out the magnetic particle that is attached to detection molecules by chemical reaction.With reference to figure 5, in whole course of reaction, be conventionally carried at the first binding molecule 5004 in detection molecules 5003 and be carried at the second binding molecule 5005 on magnetic particle 5006 between adhesion maximum, bonding state is not easy fracture.And catch adhesion between biomolecule 5001 and testing molecule 5002, testing molecule 5002 and detection molecules 5003 relatively a little less than, bonding state easily causes fracture because cleaning.Therefore, need to calibrate this impact test result being produced by the flow velocity of cleaning fluid and the variation of volume.
Fig. 8 is the fundamental diagram of the magnetosensitive biologic sensor chip of the example 3 according to the present invention.Magnetosensitive biologic sensor chip 806 comprises first area 8100 and second area 8200.The surface of first area is for example modified with biometric capture molecule 8001 as test zone, the content that the test reaction occurring on it and method of testing are described above with reference to Fig. 5.Second area surface is directly modified with the testing molecule identical with testing molecule 8002 of predetermined quantity as calibration region.Using the testing molecule of this predetermined quantity in theory or in experience by the corresponding output signal of magnetic particle that is attached in conjunction with detection molecules the reference value as this calibration region.When detection molecules 8003 is flowed through chip 806 surperficial, both with first area 8100 in testing molecule 8002 also with second area 8200 in testing molecule 8002 react.The detection molecules that is combined in chip surface is reacted with the magnetic particle of flowing through subsequently, and magnetic particle 8006 is combined in to chip surface.After having reacted, chip surface is rinsed, to clean to the magnetic particle that deposits to chip surface.By respectively first area and second area being measured, obtain the test signal of first area and the calibrating signal of second area.Reference value based on this calibration region and the calibrating signal measuring can be determined the calibration factor of this calibration region.
But the calibration factor as above obtaining has only characterized the impact of the fracture of combination between testing molecule and detection molecules on test result, and do not consider to catch in test zone the impact of the fracture of combination between molecule and testing molecule on test result.Therefore need to be on further considering through the calibration factor correction obtaining like this due to the impact of the fracture that catches combination between molecule and testing molecule on test result.Conventionally, can think that the testing molecule and the test surfaces that are directly modified on calibration region test surfaces have enough bond strengths, are not subject to the impact of cleaning fluid.Be to be understood that, when testing molecule is known with the binding ability that catches molecule, the binding ability of testing molecule and detection molecules is when known, on first area, magnetic particle is cleaned probability that liquid washes and is cleaned the ratio of the probability that liquid washes with respect to the magnetic particle on the second area that is modified with testing molecule known, can be determined by the ratio that catches molecule and the binding ability of testing molecule and the binding ability of testing molecule and detection molecules the correction factor of calibration factor.For example, when testing molecule is identical with the binding ability of testing molecule and detection molecules with the seizure binding ability of molecule, for the magnetic particle of being combined with detection molecules, the magnetic amounts of particles of losing because clean in calibration region should equal 50% of magnetic amounts of particles that test zone loses.Preferably, for the calibration region of finishing testing molecule, with described correction factor, the calibration factor obtaining is revised, can be obtained revised calibration factor.
The technical scheme of this example can be used separately, also can be combined with the technical scheme of other examples, to obtain technique effect of the present invention.
Example 4
Fig. 9 illustrates the fundamental diagram of the magnetosensitive biologic sensor chip of the example 4 according to the present invention.Magnetosensitive biologic sensor chip 906 comprises first area 9100 and second area 9200.The surface of first area is for example modified with biometric capture molecule 9001 as test zone, the content that the test reaction occurring on it and method of testing are described above with reference to Fig. 5.Second area finishing be useful on the seizure biomolecule 9001 of calibration ', should be for the seizure molecule 9001 calibrated ' be different from the seizure molecule 9001 on test zone, and do not react with testing molecule 9002 and detection molecules 9003.For the testing molecule 9002 calibrated ' can with the seizure biomolecule 9001 ' reaction bonded for calibrating, and can with the 9003 ' combination of calibration detection molecules.Should for the testing molecule 9002 calibrated ' and calibration detection molecules 9003 ' all not with testing molecule 9002 and detection molecules 9003 reaction bonded.The second binding molecule 9005 of the first binding molecule 9004 of calibration detection molecules 9003 ' carry ' can carry with magnetic particle 9006 reacts, and magnetic particle 9006 is combined in the strand of calibration testing.
In order to obtain, calibrate accurately effect, for the seizure molecule 9001 calibrated ' and chip surface area 9200 between combination stability should be identical with the combination stability catching between molecule 9001 and chip surface area 9100.For the testing molecule 9002 calibrated ' and for the seizure molecule 9001 calibrated ' reaction efficiency and adhesion should accurately simulate testing molecule 9002 and catch reaction efficiency and the adhesion between molecule 9001.Equally, calibration detection molecules 9003 ' and for the testing molecule 9002 calibrated ' reaction efficiency and adhesion accurately reaction efficiency and the adhesion of analog detection molecule 9003 and testing molecule 9002.Have feature as mentioned above for the seizure molecule calibrated, be also hereinafter referred to as in this article analog capture molecule, simulation testing molecule and analog detection molecule for testing molecule and the calibration detection molecules of calibrating.In the situation that using analog capture molecule to calibrate test result, on the one hand need to be by the analog capture molecular modification of predetermined quantity the test surfaces in calibration region, the concentration of simulating on the other hand testing molecule in reaction compartment in reactant liquor need to be known, and this calibration region has reference value correspondingly thus.By known volume comprise concentration known simulation testing molecule 9002 ' calibrating reagent evenly mix with the testing sample 9002 of known volume, injection reaction compartment.The analog detection reagent that contains analog detection molecule, the detection reagent and the label that contain for detection of the detection molecules of testing molecule are entered to reaction compartment.By respectively first area and second area being measured, obtain the test signal of first area and the calibrating signal of second area.Based on this calibration region reference value and the calibrating signal that the measures calibration factor that can determine this calibration region.The calibration factor that utilization obtains is calibrated the test signal of test zone, the test signal after can being calibrated and precisely test result.
Contain simulation testing molecule 9002 ' calibrating reagent can be liquid reagent, can be also powdered reagent.If powdered reagent, this reagent can be stored in advance as shown in Figure 5 for adding the fluid reservoir 1012 of testing sample.When the testing sample of known volume joins fluid reservoir 1012, this sample simultaneously by contain simulation testing molecule 9002 ' the powdered reagent of known weight dissolve and to obtain mix reagent.Mix reagent is injected into reaction chamber and reacts.By respectively first area and second area being measured, obtain the test signal of first area magnetic particle and the calibrating signal of second area magnetic particle.
Be to be understood that, when the seizure molecule 9001 for calibrating ', for the testing molecule 9002 calibrated ' and for the detection molecules 9003 of calibrating ' between reactive, adhesion and stability accurately when the reactivity between analog capture molecule 9001, testing molecule 9002 and detection molecules 9003, adhesion and stability, this example can be used for calibration due to difference and test environment and the condition impact that various factors brings measurement result on an equal basis of chip differences, microfluid sample boat difference, various reaction reagents, thereby improves the measuring accuracy of sensor chip.
The technical scheme of this example can be used separately, also can be used in combination with other examples, obtains technique effect of the present invention.
Example 5
Utilizing microfluid sample boat to react testing sample and reaction reagent, and when reaction result is tested, the testing sample and the various reaction reagent that participate in reaction can add respectively reaction compartment step by step, and each reactions steps that forms strand shown in Fig. 5 can complete step by step.This reactive mode is called as multistep reaction.The reaction of example 1-4 as above is multistep reaction.If the reagent of testing sample and all participations reaction is injected reaction compartment simultaneously, different molecular between reaction while synchronously carrying out, this reactive mode is called as one-step reaction.This example is modified with analog capture molecule as example by take the test surfaces of calibration region, and concrete introduction usingd one-step reaction during as method of testing, the self calibration to magnetosensitive biologic sensor chip.
Figure 10 illustrates the fundamental diagram of the magnetosensitive biologic sensor chip of the example 5 according to the present invention.Magnetosensitive biologic sensor chip 1006 comprises first area 10100 and second area 10200.The surface of first area is for example modified with and catches molecule 1 0001 as test zone.Second area finishing have the analog capture molecule 1 0001 of predetermined quantity ', this analog capture molecule 1 0001 ' be different from catches molecule 1 0001, and does not react with testing molecule 10002.During reaction, the testing sample that contains testing molecule 10002 of known volume, known volume contain concentration known simulation testing molecule 10002 ' calibrating reagent, respectively with the detection molecules 10003 of binding molecule and analog detection molecule 1 0003 ', and injected reaction compartment with the magnetic particle reagents of binding molecule simultaneously.Catch molecule 1 0001 and analog capture molecule 1 0001 ' respectively and testing molecule 10002 and simulation testing molecule 10002 ' react, detection molecules 10003, analog detection molecule 1 0003 ' also respectively with testing molecule 10002 and simulation testing molecule 10002 ' react.After having reacted, the cleaning of injection cleaning fluid is not combined in chip surface through biochemical reaction but is attached to the magnetic particle on chip surface by non-specific process, then tests.By respectively first area and second area being measured, obtain the test signal of first area and the calibrating signal of second area.The reference value that the concentration of the analog capture molecule of being modified by predetermined quantity based on this calibration region, known simulation testing molecule is definite and the calibrating signal measuring can be determined the calibration factor of this calibration region.The calibration factor that utilization obtains is calibrated the test signal of test zone, can obtain test signal and the test result accurately of calibration.
As an alternative, on the test surfaces of this calibration region, also can modify simulation testing molecule 10002 ' but not analog capture molecule 1 0001 '.In this case, need to by the testing sample that contains testing molecule 10002 of known volume, respectively with the detection molecules 10003 of binding molecule and analog detection molecule 1 0003 ', and inject reaction compartment with the magnetic particle reagents of binding molecule simultaneously.Catch molecule 1 0001 and react with testing molecule 10002, detection molecules 10003, analog detection molecule 1 0003 ' also respectively with testing molecule 10002 with simulate testing molecule 10002 ' react.To similar to the description of example 3 in the calibration of test signal and instructions, repeat no more here.
Calibration region in this example can be used alone, and also can use in conjunction with example 2.
More than describe to be to take with magnetic particle that the magneto-dependent sensor chip that is label describes as instantiation, those skilled in the art are to be understood that, technical scheme of the present invention is also suitable for using the detection system of other labels, as the Optomag detection system of Philips with use the multi-region detection system that fluorescence is label.
Obviously, above-mentioned example of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give all embodiments exhaustive.Every still row in protection scope of the present invention of apparent variation that technical scheme of the present invention extends out or change that belong to.
Claims (17)
1. a biologic sensor chip, is characterized in that,
This biologic sensor chip comprises test zone and at least one calibration region,
The test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule,
The test surfaces of each calibration region is modified with the calibration molecule of predetermined quantity and has corresponding reference value,
Described test zone has the test output of the measuring-signal of output test zone,
Each calibration region has the calibration output of the measuring-signal of this calibration region of output.
2. biologic sensor chip as claimed in claim 1, it is characterized in that, described test zone and described at least one calibration region have identical size transverse to fluid flow direction, locate described test zone and described calibration region makes, when this biologic sensor chip is placed in the reaction chamber of microfluid sample boat, the fluid flow state on regional test surfaces is identical.
3. biologic sensor chip as claimed in claim 1, it is characterized in that, described at least one calibration region comprises that its test surfaces is modified with the calibration region of the detection molecules for detection of testing molecule of predetermined quantity, its test surfaces and is modified with the calibration region of the testing molecule of predetermined quantity, the calibration region of analog capture molecule that its test surfaces is modified with predetermined quantity and at least one in the calibration region of the simulation testing molecule that its test surfaces is modified with predetermined quantity.
4. biologic sensor chip as claimed in claim 3, it is characterized in that, described at least one calibration region comprises that its test surfaces is modified with the calibration region of the testing molecule of predetermined quantity, this biologic sensor chip has correction factor, and this correction factor is determined by the ratio of the binding ability of described seizure molecule and described testing molecule and the binding ability of described testing molecule and described detection molecules.
5. biologic sensor chip as claimed in claim 1, it is characterized in that, described at least one calibration region comprises that its test surfaces is modified with the calibration region of the simulation testing molecule of predetermined quantity, this biologic sensor chip has correction factor, and this correction factor is determined by analog capture molecule and the ratio of the binding ability of simulation testing molecule and the binding ability of simulation testing molecule and analog detection molecule.
6. biologic sensor chip as claimed in claim 1, is characterized in that, described biologic sensor chip is with the serve as a mark magnetosensitive biologic sensor chip of thing of magnetic particle.
7. biologic sensor chip as claimed in claim 1, is characterized in that, the calibration molecule in described at least one calibration region has uniform surface density.
8. a microfluid sample boat, comprises biologic sensor chip as claimed in claim 1.
9. the method for testing of a biologic sensor chip, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the calibration molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
Make the testing sample that contains testing molecule enter reaction compartment,
The detection reagent that makes to contain for detection of the detection molecules of testing molecule enters reaction compartment,
Make label enter reaction compartment,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
10. the method for testing of a biologic sensor chip, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the calibration molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
Make the testing sample that contains testing molecule, the detection reagent and the label that contain for detection of the detection molecules of testing molecule enter reaction compartment simultaneously;
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
The method of testing of 11. biologic sensor chips as described in claim 9 or 10, is characterized in that, the calibration molecule in described calibration region is described molecules detected or described detection molecules.
The method of testing of 12. biologic sensor chips as described in claim 9 or 10, it is characterized in that, calibration molecule in described calibration region is testing molecule, this biologic sensor chip has the definite correction factor of ratio by the binding ability of the binding ability of described seizure molecule and described testing molecule and described testing molecule and described detection molecules
This method of testing further comprises the cleaning step before measuring process, and
With described correction factor to described calibration after test signal revise, obtain revising rear test signal.
The method of testing of 13. 1 kinds of biologic sensor chips, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the analog capture molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
The calibrating reagent of the simulation testing molecule that makes to contain concentration known and the testing sample that contains testing molecule enter reaction compartment,
The detection reagent that makes the analog detection reagent that contains analog detection molecule and contain for detection of the detection molecules of testing molecule enters reaction compartment,
Make label enter reaction compartment,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
The method of testing of 14. 1 kinds of biologic sensor chips, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the analog capture molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
The testing sample that makes to contain testing molecule, contain detection reagent for detection of the detection molecules of testing molecule, calibrating reagent, the analog detection reagent that contains analog detection molecule and the label of the simulation testing molecule that contains concentration known enter reaction compartment simultaneously
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
The method of testing of 15. biologic sensor chips as described in claim 13 or 14, is characterized in that, described in to contain the calibrating reagent of simulating testing molecule be liquid reagent or powdered reagent.
The method of testing of 16. 1 kinds of biologic sensor chips, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the simulation testing molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
Make the testing sample that contains testing molecule enter reaction compartment,
The detection reagent that makes the analog detection reagent that contains analog detection molecule and contain for detection of the detection molecules of testing molecule enters reaction compartment,
Make label enter reaction compartment,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
The method of testing of 17. 1 kinds of biologic sensor chips, this biologic sensor chip comprises test zone and at least one calibration region, the test surfaces of described test zone is modified with for catching the seizure molecule of testing molecule, the test surfaces of described calibration region is modified with the simulation testing molecule of predetermined quantity and has corresponding reference value, and the method comprises the following steps:
The testing sample that makes to contain testing molecule, contain the detection reagent for detection of the detection molecules of testing molecule, the analog detection reagent that contains analog detection molecule and label and enter reaction compartment simultaneously,
Respectively the label in test zone and the label in calibration region are measured, obtained test signal and at least one calibrating signal; And
By the calibrating signal of calibration region and the corresponding reference value of this calibration region, determine the calibration factor of this calibration region,
With determined calibration factor, described test signal is calibrated, obtain calibrating rear test signal.
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