WO2005083432A1

WO2005083432A1 - The detecting method of chip and the detecting device

Info

Publication number: WO2005083432A1
Application number: PCT/CN2004/000839
Authority: WO
Inventors: Fanglin Zou; Chunsheng Chen; Jianxia Wang
Original assignee: Chengdu Kuachang Medical Industrial Limited; Chengdu Kuachang Science & Technology Co., Ltd
Priority date: 2003-12-19
Filing date: 2004-07-21
Publication date: 2005-09-09

Abstract

The invention relates to a method for analyzing multi-reactor chip, comprises at least one of the following steps: A. increasing the viscosity of the remainder of the reaction quantitatively; B. removing and washing the remainder of the reaction simultaneously; C. weakening the background signal using the high absorbance structure out of the chip; and the step C can also be replaced by the step D: enhancing the background signal using the illuminant and/or reflecting structure out of the chip. The invention also relates to a device for analyzing said multi-reactor chip, comprises at least one of the following functional systems: A. system for increasing the viscosity of the remainder of the reaction quantitatively; B. system for removing and washing the remainder of the reaction simultaneously; C. system for weakening the background signal using the high absorbance structure out of the chip; and the system C can also be replaced by the system D: system for enhancing the background signal using the illuminant or/and reflecting structure out of the chip. The system C and the system D may be mounted at the same device and be used alternatively.

Description

Chip detection method and detection device

TECHNICAL FIELD The present invention relates to the field of detection chips, and in particular, to a method and a detection device for performing qualitative and / or quantitative analysis of a target substance in a sample, especially a biological sample, using a detection chip.

Background technique

In the present invention, a "detection chip" (also referred to as a "chip") is a detection device in a designated and / or quantitative analysis. The reactor contains multiple microprobes, and these microprobes are the same as those in the sample. The result of the specific reaction of the target molecule can be identified in an addressable manner. Chips include biochips (such as "Biochip", "Bioarray" in English) and non-biochips. Currently, the most commonly used are biochips, and the most commonly used biochips are peptide chips and gene chips. The chip includes a microchannel chip and a microarray chip (such as "Microarray" in English), but it is well known that it does not include an existing quick test reagent strip. The core of the chip is the reactor therein, and the core of the reactor is the wafer-based probe region in which the probe is fixed. In a chip reactor, the distribution density of the probes in the probe region on the substrate is greater than 10 points / cm ² . Biochip probes include all biologically active substances that can be immobilized on a solid support, such as antigens, antibodies, single- and multi-stranded DNA, RA, nucleotides, ligands, ligands, peptides, cells, Tissue components and other biological components. The chip has a wide range of applications, including gene expression detection, gene screening, drug screening, disease diagnosis and treatment, environmental monitoring and governance, and judicial identification. The chip can have different types of reactors: according to whether all samples are directed to flow on the reactor probe array when loading, the reactors are defined as flow and non-flow reactors, and the biochip is characterized by these two reactors It is defined as a mobile biochip and a non-mobile biochip; according to whether the top of the reactor probe array is open when loading, the reactor is defined as an open and non-open reactor, which are characterized by these two reactors Biochips are defined as open and non-open biochips, respectively. According to the number of reactors n on the biochip, biochips are defined as single-reactor biochips (n = 1) and multi-reactor biochips (n is equal to or greater than 2). Currently, the widest The widely used multi-reactor chip is an open-type non-flowing multi-reactor array chip with a flat chip substrate, wherein the reactor is both an open-type reactor and a non-flowing bio-chip (refer to our other patent applications: PCT / CN03 / 00055 and PCT / CN2004 / 000169).

The existing chip detection includes two types of light signal detection and non-light signal detection. Examples of the latter are the SELDI-TOF-MS method (Surface-Enhanced Laser Dissociation and Laser Ionization Time-of-Flight Mass Spectrometry, Surface-Enhanced Laser Desorption / Ionization-Time of Flight-Mass Spectra), for example, Ciphergen Corporation, USA Film-based ProteinChip Array system. Optical signal detection includes luminescent detection and non-luminescent light signal detection. Luminescence detection mainly includes fluorescence detection, chemiluminescence detection and composite light irradiation detection. The term "multi-reactor chip detection method" (hereinafter referred to as a chip detection method or detection method) in the present invention refers to a method for detecting by using a multi-reactor chip, and includes any method used in the formation, reading and analysis of a sample detection signal. One way. The formation of the sample detection signal includes sample processing, addition to the reactor, reaction, removal and washing of reaction residues, and removal and washing of optional labels and labeled residues. The reading and analysis of the detection signal includes the reduction or enhancement of the background light signal, the reading of the light signal, and the analysis in the present invention. '

The term “chip detection device” (hereinafter referred to as a detection device) in the present invention refers to a device for chip detection other than a chip, and includes a sample detection signal forming device, a detection signal reading and analysis device, and the like. The device for forming a sample detection signal refers to a device system for forming the above-mentioned sample detection signal, for example: a sample processing device, a sample adding device, a reaction device (or system), a reaction residue removal and washing device (or system), Marker adding device, marker removal and washing device, etc. The detection signal reading and analysis device includes all the device systems and instruments that perform the above-mentioned signal reading and analysis, such as: a confocal scanner, a CCD scanner, including a background light signal attenuation or / and enhancement system in the present invention, Visible light scanner, etc.

The currently used multi-reactor chip detection method, in which the method of removing reaction residues and washing the reactor is similar to the method of removing reaction residues and washing the reactor in the ELISA detection method. The device is washed, and it usually takes 3-5 minutes for each chip operation.

At present, when preparing a chip and using the prepared chip for detection, reducing background noise and increasing the signal-to-noise ratio are an important aspect of improving detection sensitivity, and involve substrate preparation There are many patents related to detection conditions and signal detection instrument preparation and detection conditions (such as U.S. Patent Nos. 6,573,048, 6,496,307, 5,827,661, 5,279,93, and 6,664,060), including the background signal dynamic attenuation during signal reading, and No background signal static reduction is performed. In the present invention, the dynamic weakening of the background signal refers to the weakening of the background signal obtained by inputting energy, for example, reducing the background noise by cooling the CCD. In the present invention, the static weakening of the background signal refers to the weakening of the background signal obtained by introducing a special material, for example, reducing the background noise by introducing a high absorbance structure.

The current chip detection methods are based on the principle of reducing background signals. Even reducing background noise has become one of the reasons why the cost of chips and signal detection instruments is currently high. In the chip detection, the substrate used in the fluorescence detection method is basically a transparent glass slide (for example, aminated, aldehyde-based, polylysine substrate), and the substrate used in the chemiluminescence detection method is basically transparent Glass slides, plastic plates or metal films (such as silver films), polymer films (such as PVDF film, nylon film, nitrocellulose film, acetate fiber film, etc.) that are basically diffusible in the substrate used in the composite light irradiation detection method. However, the current composite light irradiation detection method based on a diffusible polymer film substrate has low sensitivity; the current chemiluminescence detection method is not very sensitive; the current fluorescence detection method, although the sensitivity is higher than the previous two High, but the background noise of the slide substrate is not low, the cost of the activated substrate is high, the detection device is expensive and other shortcomings, affecting the large-scale application of the biochip method.

On the other hand, whether it is a chip or a signal detection instrument, the reduction of background noise is always limited, and sometimes even full of contradictions. For example, in order to increase the fixing amount of the probe and thereby increase the detection sensitivity of the chip, it is necessary to increase the activity or / and concentration of the surface-active groups on the substrate, but at the same time increase the area on the substrate other than the probe point. Non-specific binding activity, thus increasing the risk of increased background noise. For another example, in order to increase the intensity of a detectable signal, a laser power source with a larger power is required in the laser confocal scanner. However, it increases both the cost and the risk of photobleaching. For example, in order to increase the intensity of a detectable signal, a more powerful excitation light source is required in the CCD scanner, but it increases both the cost and the instrument noise (such as dark current).

In short, the existing chip detection methods are complicated in operation, take a long time, consume more energy, and the sensitivity needs to be improved.

The chip detection device is a material means for implementing a chip detection method. Although some chip detection devices are disclosed, especially microfluidic chip detection devices (eg, No. 01251225.7 And Chinese Patent Application No. 02261425.7), the most commonly used open-type non-flowing chips are mainly used for manual loading, reaction, sample transfer, washing and other operations, and then the chips are scanned in a laser confocal scanner or CCD scanner The image is output, and the detection results are analyzed by analysis software. Although the liquid spray method has been used for a long time in the detection of single-reactor chips, other techniques have been chosen for the automation of sample loading and washing on multi-reactor chips. For example, the reactor on the chip is designed to have the same size and the same interval as the reaction well of the microtiter plate, and the existing ELISA detection equipment is used for automatic detection (for example, Chinese Patent Application No. 01212228.8). However, since the plate washer used for the microplate is used for pumping liquid to remove the reaction residue and spray liquid to perform reactor washing, the operation time of each reactor is still long, and the pressure of the washing liquid is low, and the cleaning effect is difference.

Therefore, the development of a detection device with convenient operation, short time required, and high sensitivity, as well as a practical method, is an urgent problem in the development of chips. Summary of the invention

According to one aspect of the present invention, it provides a multi-reactor chip detection method, which is characterized by including at least one or more of the following steps: A. Quantitatively increasing the viscosity of the reaction residue; B. Simultaneously performing the reaction of the reactor Removal and washing of residues; C. Strengthening the background signal with a light-emitting or / and reflective structure not on the chip; and D. Attenuating the background signal with a high absorbance structure not on the chip; wherein the multi-reactor chip is multi- Each reactor contains an analysis chip of the same substrate. The multi-reactor chip detection method includes a detection method using the multi-reactor chip. The reaction residue refers to an unfixed reaction after the immobilization reaction in the reactor is completed. The sample and the reagents optionally present, the high absorbance structure is a structure having a signal light absorption greater than 95%, preferably greater than 97%.

According to another aspect of the present invention, a chip detection device is provided, which is characterized by including at least one or more of the following functional systems: A. A thickening system for increasing the viscosity of the reaction residue; B. Performing the Synchronous purification system for removal and washing of reaction residues in the reactor; C. Background signal enhancement system including the light-emitting or / and reflective structure; and D. Background signal attenuation system including the high absorbance structure. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the relationship between various systems in a detection device according to an embodiment of the present invention. Department's schematic diagram;

FIG. 2 is a schematic diagram showing the relationship between various systems in a detection device according to another embodiment of the present invention;

FIG. 3 is a perspective view of one embodiment of a showerhead included in a synchronous purification system; FIG. 4 is a perspective view of another embodiment of a showerhead included in a synchronous purification system; and

Fig. 5 is a perspective view of still another embodiment of a showerhead included in a synchronous purification system. Specific implementation plan

the term

The term "reactor" in the present invention refers to the place where the probe specifically reacts with the target and other related structures communicating with it, such as the reaction cell in the open multi-reactor biochip and the related isolation structure and the inlet and outlet liquid structure .

The term "chip flat substrate" in the present invention, referred to as a substrate, refers to a planar solid-phase carrier used to fix probes and other auxiliary agents (if any) in a chip. Its surface chemical and optical properties affect the chip. Important factors in performance and cost. The current substrate is selected from modified or unmodified glass, plastic, and metal. It can be used as the substrate, substrate or chip probe board of the present invention, for example, an activated glass slide containing one or more of the following derivatized groups: amino, epoxy, aldehyde, acyl Hydrazine (-CO-NHNH ₂ ), semicarbazide (H ₂ N-NH-CONH-), diethylaminoethyl (DEAE-), diethylmono (2-hydroxypropyl) aminoethyl (QAE -), Carboxymethyl (CM-), sulfopropyl (SP-), mercaptoethylpyridyl (MEP-), siloxane, and thiol.

The term "probe" in the present invention refers to a substance, such as an antigen, an antibody, a nucleic acid, and the like, which is immobilized on a solid-phase carrier to identify a target substance in a sample.

The term “background signal enhancement” in the present invention means that the background detection signal value is increased, for example, the color of the background detection signal is increased by introducing a colored substance into the substrate, the substrate surface, or / and the substrate back surface. The term "reduce background signal" in the present invention means to decrease the value of the background detection signal.

The term “coating” in the present invention refers to a dry film having a thickness of less than 1 mm formed by coating a solid phase material with a coating material. The term "film" in the present invention refers to a non-porous or perforated planar material having a thickness of less than 0.3 mm.

The term "sheet" in the present invention refers to a non-porous or perforated planar material having a thickness of 0.3 mm or more.

The term "labeling substance" in the present invention refers to a substance used for labeling a probe or a probe capture substance to obtain a positive result, for example, a labeling substance fluorescein contained in a common label in chip fluorescence detection.

In the present invention, the term "coating material" refers to a material having a thickness of less than 1 mm, which can obtain a specific function after being applied to a film-based substrate; and "colored coating material" refers to a coating material containing colored pigments, such as various colored paints and Various inks.

The term "target" in the present invention refers to the entirety of all substances related to the detection signal at the probe point in the chip reactor when the signal is detected, for example, including a probe, a target (if any) captured by the probe, and a marker. (If any), in addition to the probe, a probe carrier (such as a nanocarrier) and a dye, a colored pigment, and a colorant of the present invention may be provided on the probe point. In the present invention, the term "negative target" refers to the entirety of all substances related to the detection signal at the probe point in the chip reactor when a negative sample is used as the detection sample.

The term "background" in the present invention refers to the entirety of all substances related to the detected signal in the detection area surrounding the target in the chip reactor when the signal is detected, for example, except the substrate and the sealed object fixed on the substrate. , Markers, etc., may also include the backing of the film base (especially in the case of transparent film base). The substrate backing can exist on the back of the chip in one or more of the following forms when the chip is reading signals: attached to the transparent substrate, as an independent component, attached to the chip holder of the signal reader.

The term "signal-background ratio" in the present invention refers to the ratio c = a / b of the signal strength a of the target to the signal strength b of the background. The term "weak target signal-background ratio" in the present invention refers to the ratio c = al / b of the signal strength al of the weak target to the signal strength b of the background.

The purpose of the present invention is to provide a detection method with convenient operation, short time required, and high sensitivity, and a corresponding detection device for multi-reactor chip detection.

The detection method of the present invention includes a method in a process of forming a detection signal and a process of reading the detection signal. In order to achieve the purpose of the present invention, the detection method of the present invention includes a method for purifying the background of the chip which is more efficient in the process of forming the detection signal or / and a method of increasing or decreasing the background signal of the chip in the process of reading the detection signal. A multi-reactor chip detection method according to the present invention is characterized by including at least one of the following steps: A. Quantitatively increasing the viscosity of the reaction residue; B. Simultaneously removing and washing the reaction residue of the reactor; C. Strengthening the background signal with a light-emitting or / and reflective structure that is not on the chip; and D. Attenuating the background signal with a high-absorbance structure that is not on the chip; wherein the multi-reactor chip is a multi-reactor chip containing the same sheet The multi-reactor chip detection method includes a detection method using the multi-reactor chip, and the reaction residue refers to an unfixed sample and optionally existing after the immobilization reaction in the reactor is completed. Reagent, the high absorbance structure is a structure having a signal light absorbance greater than 95%, preferably greater than 97%. Although the method of the present invention, particularly the method comprising steps A or / and B, may be suitable for multiple detection modes (such as optical signal detection and non-optical signal detection) and multiple multi-reactor chips, the preferred detection is The mode is optical signal detection (especially in the method comprising step C or D). The preferred multi-reactor chip is an open non-flowing multi-reactor array chip based on a planar substrate, and more preferably an open-type non-flowing A high-reactor density array chip is an open non-flowing multi-reactor array chip with a reactor density of more than 0.5 per cm ² , preferably more than 1 per cm ² .

At present, the removal and washing of the reaction residues of the multi-reactor are performed by a step-by-step method, that is, the reaction residues are removed first, and then the washing liquid is added to scrub the reactor, and then the washing liquid is removed. The washing solution is added and removed, usually several times. For example, a manual method using a pipette, and a mechanical method using an ELISA plate washer. This discontinuous method is complicated and takes a long time (usually more than 3 minutes per chip). In addition, the purification effect of the manual method is not stable, and there is always a considerable proportion of residual liquid when the liquid is removed by the mechanical method. In the present invention, the removal and washing of the reaction residues of the multi-reactor are performed by a synchronous method, that is, the removal and washing of the reaction residues of the multi-reactor are performed at the same time, for example, using a fluid to directly flush the reaction residues and flush the reactor. (Hereinafter referred to as the fluid flushing method), and continuously dilute and remove the reaction residue using the balance of the in and out liquid (hereinafter referred to as the continuous dilution method). Through the examples of the present invention, we have found that the use of the synchronization method of the present invention overcomes the shortcomings of the existing methods, such as the complexity of the operation and the long time required, and the purification effect of the reactor is good for the improvement of sensitivity. When it is necessary to add a labeling substance for the labeling reaction during the detection, the method for removing and washing the reaction residues of the multi-reactor of the present invention can also be used for the removal and washing of the labeling reaction residues of the multi-reactor. In the present invention, the labeling reaction residue refers to a labeling substance which is not fixed after the labeling reaction in the reactor and an optional reagent. Since the reaction residues are currently removed from the reactor by suction, and the suction is based on the fluidity of the sucked material, the existing methods do not want to increase the viscosity of the residues, for example, it is not required to quantitatively reduce the volume of the reaction residues. Although the volume reduction of the liquid medium will occur during a certain period of time during heating (for example, 37 ° F), it is completely different from the quantitative reduction controlled in the present invention. Through the embodiments of the present invention, we have found that reducing the volume of the reaction residues and / or increasing the viscosity of the reaction residues can reduce the sputtering range of the spray liquid when it encounters the reactor, thereby reducing cross-contamination between the reactors. risk. In addition, the use of thickeners (such as dry powder) to reduce the reaction residues or even to lose fluidity is also beneficial to reduce the risk of cross-contamination between reactors.

In another of our inventions (International Patent Application No. PCT / CN2003 / 01009), the color difference between the background and the target in the chip reactor is maximized by coloring the chip during the chip preparation process to achieve The purpose of improving detection sensitivity. Through the embodiments of the present invention, we find that the same purpose can be achieved by weakening the background signal or strengthening the background signal through a structure other than a chip, such as a prefabricated structure in a detection device.

In current detection methods, it is the consensus of researchers to reduce the background signal (sometimes called noise) to improve detection sensitivity (for example, European patent application EP A1 1279960, US patent application US A1 20030032040, Chinese patent application CN A 1443854). And we surprisingly found through the embodiments of the present invention that the detection sensitivity can also be improved by raising the background signal sufficiently high (for example, the weak target signal-background ratio is less than 0.25). The light emitting structure includes a paint, a film, and / or a sheet containing a luminescent agent. The luminescent agent is selected from one or more of the following substances that can emit signal light: an excited luminescent substance including a fluorescent substance and an autonomous luminescent substance including a chemiluminescent substance and an electroluminescent substance.

In current detection methods, the reduction of background signals is usually performed by using low background signal chips or / and reducing dark current (such as CCD). The method for performing background signal attenuation by using the high absorbance structure in the detection device of the present invention is a method for static background signal attenuation. The high absorbance structure includes a coating, a film, and / or a sheet having an absorbance of signal light of more than 95%, preferably more than 97% (or a reflectance of less than 5%, preferably less than 3%).

An example of the detection method of the present invention containing the three steps (A, B, C or D) is as follows: (1) input the prepared sample (for example, a sample with a diluted solution, a sample with a labeled substance, etc.) into multiple reactors of the chip;

(2) The sample is reacted in the reactor under the reaction conditions;

(3) after the reaction is completed, the reaction residue is thickened to reduce or even lose its fluidity;

(4) removing and washing the reaction residue of the reactor at the same time to purify the reactor;

(5) If necessary, input a marker into the purified reaction to mark the reaction result, and after the labeling reaction, remove and wash the residue of the labeling reaction of the reactor to clean the reactor;

(6) The background signal is strengthened (step C) or the background signal is weakened (step D). The background signal of the chip is increased or decreased to read the detection signal formed in the purified chip reactor;

(7) Analyze the detection signal.

The detection method of the present invention may include only one or two steps of the present invention in addition to some other steps. For example, a chip detection method including other steps and the above steps A and 8, human and, and D, B and C, B and D, or C and D. For another example, the chip detection method includes other steps and the above steps, B, C, or D. The detection method including a plurality of the steps may enable the steps with different advantages to be used in a concentrated manner, and has more advantages.

In the multi-reactor chip detection method according to the present invention, the removal and washing of the reaction residues of the reactor are performed simultaneously under the action of a fluid. Although the simultaneous method for removing and washing the reaction residue of the present invention includes other methods (such as the above-mentioned serial dilution method), a preferred method of the present invention is a fluid flushing method. In the present invention, the fluid may be a gaseous, solid or liquid fluid. In the embodiment of the present invention, the preferred fluid is a liquid fluid, especially a liquid fluid with mechanical energy (pressure energy or / and ultrasonic energy). Although liquids containing ultrasonic energy have been used for chip washing, simultaneous removal and washing of reaction residues from the reactor using fluids containing ultrasonic energy are carried out by the present invention.

In a preferred embodiment of the invention, said removing and washing are performed using a fluid containing pressure energy. The pressure energy can be applied to the fluid with a momentum [mass X flow rate (m Xv)] to achieve the purpose of removing and washing the reaction residues of the reactor simultaneously. The above pressure can be maintained or changed during the removal of reaction residues and washing (such as continuous Or pulse changes). Through the method in the embodiment of the present invention, we find that a larger pressure is not only beneficial for reducing the cross-contamination, but also for rapid purification of the reaction residue. Of course, the fluid also includes a pressure / ultrasonic fluid having the hydraulic pressure and ultrasonic waves imparted by an ultrasonic transducer.

In the method according to the present invention, the fluid used is a liquid, and its hydraulic pressure is 0.1 to 10.0 kg / cm, preferably 1.5 to 5.0 kg / cm ² , and more preferably 2.0 to 4.0 kg / cm ² . In the embodiment of the present invention, the hydraulic pressure refers to the hydraulic pressure at the nozzle outlet. Until now, one principle to avoid cross-contamination between reactors is that the samples in one reactor and their reaction residues and washing liquid must never enter the other reactor. Therefore, in the single-reactor chip detection, liquid spraying can be used to simultaneously remove and wash the reaction residues; in the multi-reactor chip detection, no one has developed a similar method. Through the examples of the present invention, we were surprised to find that if the fluid flushing method of the present invention is used, the reaction residues of multiple reactors are simultaneously removed and washed, even if the reaction residues of one reactor or / and the washing solution enter another The reactors did not cause cross-contamination between the reactors. Moreover, because the reaction residues are removed from the reactor faster and more thoroughly, not only is the operation convenient and the operation time shortened (usually less than 45 seconds), but also the washing effect is better (for example, the number of remaining non-specific marker points is reduced) can be guaranteed Higher sensitivity.

Although it is not intended to enter the theoretical discussion, we believe that when the hydraulic pressure is appropriately high, the outlet A on the same nozzle is ejected to a reactor A, especially the fluid A on its probe array, and the adjacent outlet B is sprayed. For the fluids flowing out of the adjacent reactor B, the spattered streams formed by them, even for entering into another reactor, are difficult to enter into the probe array in the other jet region due to hydrodynamic reasons. In addition, it is assumed that the reaction A very small amount of sputtered stream a from reactor A enters reactor B, and there is a very short-term contact between the trace of highly diluted reaction residue and the liquid flow on the surface of reactor B ml / 0.16 mm ² / min), it is difficult to form a substantial reaction due to chemical reaction kinetics.

The above-mentioned methods relating to removal and washing of the reaction residues of the multi-reactor can also be used for removal and washing of the labeled reaction residues of the multi-reactor.

In the multi-reactor chip detection method according to the present invention, the viscosity of the reaction residue is increased by 10% or more, and preferably 20% or more. The current chip detection method, in which the reaction residues need to be removed first, requires a large volume of reaction residues. The method includes the step of reducing the volume of the sample or the reaction residue, thereby increasing the viscosity of the sample. Through the method in the embodiment of the present invention, we found that appropriately increasing the viscosity of the reaction residue (for example, increasing by more than 30%), or even losing fluidity, can better control the sputtering phenomenon in the fluid flushing method. Conducive to reducing the risk of cross-contamination. In addition to increasing the viscosity of the present invention, in addition to quantitative reduction in volume (physical thickening), it can also be performed in other ways, for example, by adding a tackifier (such as dry powder) to the reaction residue to increase the viscosity, or even It loses fluidity (components thicken). The range of volumetric reduction is usually 10-60%, preferably 25-45%. The thickening dry powder in the method of the present invention is a powdery substance that is soluble or insoluble and has the function of reducing liquid flowability, such as gel, water-soluble organic matter, ultrafine powder and the like.

In the multi-reactor chip detection method according to the present invention, the background signal is strengthened to more than 200%, preferably more than 500% of a negative target signal, wherein the negative target signal is a detection signal obtained using a negative sample. It is the consensus of researchers in the chip field to improve detection sensitivity by reducing the background signal. Through the embodiments of the present invention, we have surprisingly found that the detection method for enhancing the background signal of the present invention can significantly improve the detection sensitivity (refer to the international patent application with application number PCT / CN2003 / 01009). On the other hand, the background signal can also be reduced to less than 80%, preferably less than 70%, of the negative target signal. In current chip detection methods, the negative target signal is higher than or similar to the background signal.

Both the background signal enhancement method and the background signal attenuation method of the present invention are aimed at maximizing the signal contrast between the target and the background. In fact, the signal contrast between the target and the background is a sign of detectability. For example, for infrared stealth technology, Maclean et al. Used the contrast ratio radiation C to represent the thermal imager's detectability: C = Eo-Eb. In the formula, Eo is the target specific emissivity, Eb is the background specific radiance, and Eo and Eb are directly proportional to the absorption of the target material and the background material, respectively. For infrared stealth, the larger the C, the higher the resolution and the greater the detectability of the camera. Conversely, C is in the best state of stealth when it approaches zero. Stealth technology aims to minimize the contrast between the target and the background, and the technology of the present invention aims to maximize the contrast between the target and the background. Therefore, the research of the present invention has selected "obviousness" as the technical basis. The technology of the present invention can be referred to as the manifestation technology, and the detection method of the present invention can be referred to as the manifestation technology detection method.

According to the present invention, there is also provided a chip detection device, which can be used in the above-mentioned multi-reactor chip detection method of the present invention, and includes at least one of the following functional systems: A. Performing the reaction A thickening system that should increase the viscosity of the residue; B. a simultaneous purification system for removing and washing the reaction residue of the reactor; C. a background signal enhancement system containing the light-emitting or / and reflective structure; and D. containing The background signal attenuation system of the high absorbance structure. In addition to the above-mentioned functional system, the detection device of the present invention includes other systems that enable the detection to be completed, such as: sample preparation systems, sample input systems, reaction systems, marker preparation systems, marker input systems, detection signal reading and analysis System, etc. Like many other detection devices, different functional systems can include different devices and instruments, and can also include some of the same devices and instruments. For example, the above-mentioned synchronous purification system for removal and washing of reaction residues (referred to as the synchronous purification system for reaction residues) may include some of the same devices and instruments as the synchronous purification system for removal and washing of labeled residues (referred to as the synchronous purification system for labeled residues). (Such as pumps and sprinklers).

The detection device of the present invention, particularly the detection device including the functional system A or / and B, may be an optical signal detection device or a non-optical signal detection device. The preferred detection device is an optical signal detection device. The detection device of the present invention can use different multi-reactor chips, but the preferred detection device uses an open non-flow multi-reactor array chip based on a flat substrate, especially an open non-flow high-reactor density array chip. In the chip luminescence detection device or one of the functional systems, one chip can be operated at a time, or multiple chips can be operated simultaneously; part of the reactors on one chip can be operated at the same time, Operate all reactors on one chip.

In the detection device of the present invention, the viscosity-increasing system (functional system A) has the purpose of reducing cross-contamination and wind drop. The purpose of the solution is to reduce or even lose the fluidity of the reaction residue. The principle of solution realization is to improve the reaction. The viscosity of the residue can reduce its fluidity; the synchronous purification system (functional system B) aims to improve the purification efficiency of the reactor and avoid cross-contamination. The purpose of the solution is to remove and wash the reaction residue of the reactor at the same time. The principle of solution realization is that at the same time cleaning can be performed continuously with a larger volume of cleaning agent; the background signal enhancement system (functional system C), whose purpose is to improve detection sensitivity, and the purpose to achieve the solution is to introduce the light emission Or / and the reflective structure raises the background signal of the chip sufficiently high, and the principle of the solution is that raising the background signal of the chip sufficiently high can increase the signal contrast ratio between the negative target and the limit weak positive target and the background, thereby improving the detection sensitivity; Background signal attenuation system (functional system D), the purpose of which is to improve detection sensitivity, The solution is introduced into the realization of a high absorbance to reduce core structure The background principle of the solution is that the background signal of the chip is reduced enough to increase the signal contrast ratio between the negative target and the limit weak positive target and the background, thereby improving the detection sensitivity. .

The functional systems in the detection device of the present invention have a clear logical relationship. Referring to Figures 1 and 2, a chip detection device of the present invention containing the functional systems A, B, C, and D. An example of the operation steps and the logical relationship between the systems is as follows:

(1) Use a sample input system (such as a pipette or a sampler with a micropump) to input the sample prepared by the sample preparation system (such as a sample with diluted solution, a sample with labeled substance, etc.) into the chip for multiple reactions Device

(2) The reaction conditions of the sample in the reactor are provided by a reaction system (such as a reaction system containing a chip incubator and a temperature / humidity controller);

(3) After the reaction is completed, the viscosity-increasing system (functional system A) is started to reduce the fluidity of the reaction residue or even lose it;

(4) Restart the synchronous purification system (function system B) of the reaction residues and simultaneously remove and wash the reaction residues of the reactor;

(5) If necessary, use a marker input system (such as a pipette or a sampler containing a micropump) to input the marker prepared by the marker preparation system into multiple reactors of the chip after washing and drying. The system provides the conditions for the labeling reaction. After the reaction, the labeling residue synchronous purification system (which can be the functional system B) is used to simultaneously remove and wash the labeling reaction residue of the reactor (Figure 2);

(6) The cleaned and dried chip is sent to the scanner, and the background signal enhancement system (functional system C) or background signal attenuation system (functional system D) is selected to increase or decrease the background signal of the chip according to the detection method;

(7) The detection signal reading and analysis system is used to read and analyze the detection signal in the chip reactor.

The detection device of the present invention may also include only one, two, or three of the functional systems and some other systems. For example, only the detection devices of the functional system 8, C or D and some other systems are included. As another example, the chip detection device includes the functional system person and 8, BC, B and D, C and D, respectively. For another example, the chip detection devices include the functional systems A, B and CA, B and D, and B, C, and D, respectively. Generally speaking, the more the functional system is included, the higher the efficiency of the chip detection device is. This invention contains only these In a chip detection device of a functional system, the above-mentioned logical relationship is basically unchanged, except where the functional system (functional systems A, B, C, D) of the detection device of the present invention is not included, or other corresponding functional systems are replaced [eg Step-by-step washer is used to replace functional system B, and a scanner that does not include the signal enhancement system (functional system C) or background signal attenuation system (functional system D) is used instead of the signal enhancement system (functional system C) or Scanner for background signal reduction system (functional system D)], or cancel these systems (such as functional system A).

The detection device of the present invention may be a complete detection device capable of performing all detection steps (for example, the above steps (1) to (7)), and thus including all detection function systems, or may perform only a part of the detection steps (for example, the above step (3)) And (4), or (3) to (7)), so that only a part of the detection function system is included. The detection device of the present invention including a plurality of detection function systems includes a central control system for controlling the relationship of each detection function system. In addition, the same device may be used for different functional systems. For example, in one embodiment of the present invention, the above-mentioned synchronous cleaning system device is used for both the reaction residue cleaning system and the marking residue cleaning system.

In the chip detection device according to the present invention, the synchronous purification system includes a pump and a spray head, wherein the pump provides a flow rate and a hydraulic pressure of the fluid, and the spray head distributes the fluid to a plurality of reactors of the chip . The synchronous purification system may further include the following components: A. the flow direction control mechanism of the outlet; B. the relative position control mechanism of the outlet and the reaction surface of the reactor (such as a rotatable chip holder), C. ultrasonic Transducer, etc. It may also include a sealed chamber that prevents the pressure fluid from contaminating the outside world.

The pump contains hydraulic and flow rate control systems. The spray head includes a liquid inlet 1 and a liquid outlet 2, where the liquid outlet 2 may have different types, for example, as shown in FIG. 3, the convex nozzle of the liquid port 2 on the top of the convex body, as shown in FIG. A concave nozzle with a liquid outlet at the bottom of the concave body, a surface nozzle with the liquid outlet 2 distributed on a plane as shown in FIG. 5, and a combination thereof. During spray washing, the liquid flow may be pulsed, continuous, or a combination thereof. The nozzle and / or chip position can be moved mechanically, such as circularly or oscillatingly, to make spraying more uniform.

The reaction residue removal and washing system can also be used as a label residue removal and washing system. Marking residue removal and washing systems can also be performed using other methods, such as single-port pressure fluidizers, single-row nozzle pressure fluidizers, and so on.

Through the examples of the present invention, we have found that by using the purification system of the present invention to simultaneously remove and wash reaction residues, not only has not been reacted in multi-reactor chips, even high-density Cross-contamination on the core and a higher cleaning effect.

In the chip picking and testing device according to the present invention, a plurality of outlets for fluids may be provided on the spray head, and the number and density of the outlets are not less than the number and density of the reactors of the multi-reactor chip. The removal and washing of reaction residues on any of the reactors in the chip is performed by the fluid from one or more of the outlets. A preferred fluid is the fluid containing pressure energy (the fluid is referred to as a pressure fluid in the invention, and its hydraulic pressure at the outlet is 0.1-10.0 kg / cm ² , preferably 1.5-5.0 kg / cm ² , more preferably 2.0-4.0 kg / cm ² ) o

In the chip detection device according to the present invention, the synchronous purification system has one or more of the following operating parameters: A. The outlet density is greater than 0.5 / cm ² , preferably greater than 1 / cm ² ; B. The fluid The clockwise angle between the direction of the liquid flow at the outlet and the substrate base plane of the fixed probe of the chip is between 5 and 350 degrees, preferably 90 ± 5 degrees or 180 ± 5 degrees; C. The outlet and The distance between the substrate planes of the reactor is between 0.1 and 10.0 cm, preferably between 1 and 3 cm; D. The working hydraulic pressure of the outlet is 0.1 to 10.0 kg / cm ² , preferably 1.5 to 5.0 kg / cm ² And more preferably 2.0 to 4.0 kg / cm ² . In addition, the outlet diameter, spray speed, etc. are also important working parameters, which are related to the probe array size, probe array spacing, and other reactor design data, and will not be listed one by one. In summary, reducing the risk of cross-contamination is the main determinant of these operating parameters.

The clockwise angle between the direction of the liquid flow at the outlet and the reaction surface of the reactor where the reaction residue is located can be adjusted by an angle adjuster provided inside or outside the purification system or manually adjusting the spray head or / And the horizontal angle of the chip. The preferred angle is from 90 to 5 degrees when the flow direction is from low to high (spray upwards), and at 180 ± 5 degrees, the liquid direction is from high to low (spray downwards).

The distance between the outlet and the reaction surface of the reactor can be adjusted by lowering or raising the shower head or chip.

In our study, no cross-contamination was observed under the above operating parameters. In the chip detection device according to the present invention, the viscosity increasing system includes a temperature controller or / and a humidity controller. The temperature or / and humidity control is preferably a humidity control.

In the chip detection device according to the present invention, the tackifier system contains a tackifier transporting device.

In the chip detection device according to the present invention, the background signal enhancement system includes Parts and components of coatings that emit light signals. Wherein, the coating is the functional basis of the background signal enhancement system, and is distributed in one or more places in the detection device that can affect the background signal of the chip, such as the chip carrier and the vicinity of the chip carrier during signal reading. component. In the embodiment of the present invention, the coating of the coating includes white paint and fluorescent whitening paint.

In the chip detection device according to the present invention, the background signal reduction system includes parts and components including a coating for detecting light reflectance of less than 3%. Wherein, the coating is the functional basis of the background signal attenuation system, and is distributed in one or more places in the detection device that can affect the background signal of the chip, such as the chip carrier and the vicinity of the chip carrier during signal reading. component. In the embodiment of the present invention, the paint of the coating layer includes a black paint for optical use and a matte black paint processed by nanotechnology. The surface roughness Ra of the coating is between 0.2 and 3 μm, preferably between 0.5 and 2 μm. Examples

Example 1: Preparation of a chip detection device containing a synchronous purification system

The chip detection device prepared in this embodiment includes three parts: a sample detection signal forming device, a detection signal reading device, and a detection signal analysis device. In this embodiment, the sample detection signal forming device includes a reaction system, a multi-reactor residue removal and washing system, a chip drying system, and a chip transportation system; the detection signal reading device includes a chip transportation system and a signal scanning system; and detection signal analysis The device includes a computer, computer software, and a printer. In addition, the chip detection device also includes a chip transport system and a central control system that handle the logical relationships and connections between the devices. The chip detection device of this embodiment can also be easily configured with a reaction medium processing and adding system, a reaction reagent storage system, and the like.

The chip detection device prepared in this embodiment may be a light-emitting chip detection device (such as a detection device based on a fluorescent substance label), or a non-light-emitting chip detection device, such as a gold label-silver amplification detection method. Chip detection device (for gold mark-silver amplification detection method, refer to EP1179180 A1).

The first synchronous purification system prepared in this embodiment is a continuous dilution purification system, which includes an input liquid pump, an output liquid pump, and an input liquid and output liquid flow balance control system. The working principle is: based on the continuous dilution method, the reaction residue is continuously diluted and removed by using the balance of the in and out liquid. Compared with the existing distributed cleaning and washing machine, the continuous dilution and purification system of this embodiment overcomes the weakness that liquid absorption always has residual liquid, and implements continuous dilution / washing to reduce the net 化时间。 Time.

The second synchronous purification system prepared in this embodiment is a synchronous cleaning system for pressure fluid. It contains at least three subsystems: pressure fluid generation and transportation systems (such as wash liquid storage bottles, pressure pumps, pipes, multiple nozzles, etc.); pressure fluid application control systems (such as pressure / flow rate controllers, wash temperature Relative position control devices such as the angle of control, the nozzle outlet and the substrate plane of the chip fixed probe, etc .; waste liquid discharge and antifouling systems (such as the drain channel, waste liquid tank, an openable and closed Isolate the washing room, etc.). Its working principle is: the pressure pump sprays the washing liquid in the washing liquid storage bottle to the probe arrays of the multiple reactors of the chip at a preferred pressure and through multiple nozzle outlets at a preferred angle and distance to achieve a cross-contamination Residuals in the simultaneous multi-reactor were removed and washed. In the purification system II prepared in this embodiment, some of the operating parameters are adjusted by the following devices: A. Four detachable and replaceable multi-nozzle heads (all containing circular outlets, see Table 1); B. Make the pressure fluid outlet The angle adjuster whose clockwise angle between the direction of the liquid flow and the substrate plane of the chip's fixed probe varies between 5 and 350 degrees; C. The distance between the pressure fluid outlet and the substrate plane is 0.1- Distance regulator with a change between 5.0 cm; D. Pressure regulator with a working fluid of pressure fluid at the outlet that changes between 0.1 -5.0 kg / cm ² . Compared with the above-mentioned continuous dilution purification system, the pressure fluid synchronous purification system of this embodiment does not need to be balanced in and out of the liquid. In particular, high-pressure fluid washing can be performed to improve purification efficiency. Table 1. Multi-nozzle parameters

The third synchronous purification system prepared in this embodiment is a pressure / ultrasonic fluid synchronous purification system. It is made by adding an ultrasonic transducer in the pipeline of the pressure pump and the nozzle outlet in the pressure fluid synchronous purification system. Its working principle is similar to the working principle of the above-mentioned pressure fluid synchronous purification system, except that the washing liquid is energized by an ultrasonic transducer in addition to energy by a pressure pump. The pressure / ultrasonic fluid synchronization net of this embodiment The chemical system is particularly suitable for the detection of chips that are difficult to wash.

In this embodiment, when the systems are arranged in a straight line in the chassis, they can be arranged on the side of the chip transport system. After the chip completes one step operation of a system, it is sent to the chip transport system and then sent to the corresponding position of the next operation step for the next operation. The systems can be combined in a fixed form or in the form of independent units. When the systems are arranged in a ring shape in the chassis, the transportation system is a disk, and the systems are arranged on or around the transportation disk system. After the chip completes the operation steps of a system, the disk rotates, and the chip is transported to The next operation is performed at the operating position on or around the next disc. When the chip is ready to be scanned after it is dried, another transporting part sends the chip to the scanning position for scanning. The systems are grouped together in a fixed form.

The optional chip drying system consists of a blower and duct. There can be one or more air outlets.

The chip detection device prepared in this embodiment may be an automatic detection device, a semi-automatic detection device, or a manual detection device.

The synchronous purification system prepared in this embodiment can also be used alone, for example, in the form of a synchronous purifier. Example 2: Preparation of a chip detection device containing a thickening system and a synchronous purification system

The chip detection device prepared in this embodiment is similar to the chip detection device prepared in this embodiment 1, except that it includes the above-mentioned synchronous purification system, and the sample detection signal forming device also includes a thickening system.

The first tackifier system prepared in this embodiment is a component tackifier system, that is, tackifiers are added to the medium to be thickened (for example, the reaction residue solution) to increase the viscosity. The thickener in this embodiment includes dry powder monosaccharide, polysaccharide or polysaccharide. The component thickening system in this embodiment includes a thickener storage bottle, a pipeline, a pump, a spray head containing a thickener outlet, and a control system for the amount and method of adding thickener. Its working principle is: use a pump to add the thickener through the nozzle outlet, and add it to the reaction residue of the chip reactor in a preferred amount and manner to increase the viscosity to reduce the risk of the reaction residue entering another reactor or / And reduce the risk of non-demand response, thereby reducing the risk of cross-contamination. In addition,

The second thickening system prepared in this embodiment is a physical thickening system, that is, by increasing the concentration of the thickening medium (such as reaction residues) (without increasing the component conditions) (By reducing the volume) to increase viscosity. The physical viscosity increasing system in this embodiment includes a temperature / humidity controller. The working principle of the physical thickening system in this embodiment is: by adjusting the temperature, humidity, and time, the evaporation amount of the liquid (such as water) in the medium to be thickened is controlled, and the thickening is performed by concentration to reduce the residual reaction The risk of substances entering another reactor reduces the risk of cross-contamination.

The thickening system prepared in this embodiment can also be used alone, for example, in the form of a thickener. The thickening system in this embodiment can also be used independently in combination with the synchronous purification system. Example 3: Preparation of a chip detection device including a viscosity increasing system, a synchronous purification system, and a background signal attenuation system

The chip detection device prepared in this embodiment is similar to the chip detection device prepared in this embodiment 2, but it also includes a background signal attenuation system in the sample detection signal reading device.

The background signal attenuation system prepared in this embodiment includes a highly absorbing black paint coated on a chip holder and a light source, optical components of a light background signal detection portion, and a surface that can cause light reflection around. Its working principle is: When scanning the signal light, the light transmitted through the background of the chip (preferably a transparent chip) is absorbed by the highly absorbing black coating on the chip holder, which reduces the reflectance and thus the intensity of the background signal; The black coating on other components helps reduce background noise by absorbing various interfering light (such as scattered light). In this embodiment, the sample detection signal reading device is a confocal laser scanner with fluorescence excitation light (540 nm) and fluorescence emission light (570 nm). In this embodiment, the paints used are three kinds of black high-absorptive paints used for the inner walls of telescopes and camera lenses. These coatings contain ferrite and silicon oxide, and the surface of the coating formed has a certain roughness (surface roughness Ra between 0.02 and 3 m), and the absorbance of the formed coating is 91%, 95%, and 97, respectively. %. The higher the absorbance of the coating used, the better.

The background signal attenuation system prepared in this embodiment can be used independently, for example, in the form of a confocal laser scanner or a laser scanner containing the above-mentioned high absorbance coating. Example 4: Preparation of a chip detection device including a viscosity increasing system, a synchronous purification system, a background signal reduction system and a background signal enhancement system The chip detection device prepared in this embodiment is similar to the light-emitting chip detection device prepared in this embodiment 3, except that it includes a background signal attenuation system, and it also includes a background signal enhancement system in the sample detection signal reading device.

The background signal enhancement system prepared in this embodiment includes a light emitting structure covered on a surface of a chip holder in a confocal laser scanner. The working principle is as follows: When scanning the signal light, the signal light on the background of the chip (preferably a transparent chip) is enhanced by the light-emitting structure on the chip holder, which improves it with low signal targets (such as negative samples and extreme weak positive samples). Detection signal), thereby improving detection sensitivity. The light-emitting structures used in this embodiment are a polyethylene film containing a fluorescent whitening agent and a white paint containing a fluorescent whitening agent. The surface of the white paint coating has a certain roughness (surface roughness Ra is between 0.02 and 3 m).

In the background signal enhancement system prepared in this embodiment, in the same detection device as the background signal attenuation system, the chip holder is replaceable. When the background signal attenuation is needed, the scanner uses the above-mentioned high absorbance coating. Layer chip holder; when background signal enhancement is needed, the scanner uses the chip holder containing the above-mentioned reflective structure.

The background signal enhancement system prepared in this embodiment can also be used independently, for example, in the form of a confocal laser scanner or laser scanner containing the above-mentioned light emitting structure. It can also be used in a scanner (such as a confocal laser scanner or laser scanner) with the background signal reduction system described above. Example 5: Chip detection method with synchronous purification

The simultaneous purification method in this embodiment refers to simultaneous removal and washing of reaction residues.

The chip used in this embodiment is a high-hydrophobic isolation structure analysis chip manufactured according to another invention (PCT / CN2004 / 000169). The high-hydrophobic isolation structure is a strip-shaped high-hydrophobic convex body having a height of 25-750 | Lim and a width of 2.0-2.5 mm. The substrate pool surrounded by the high-hydrophobic convex body is a 3 mm × 3 mm rectangle. There are a total of 14 substrate cells on one epoxy glass slide (75 X 25 X 1.0 mm) in the horizontal direction, 4 substrate cells in the vertical direction, and 56 substrate cells in total. The probes fixed in the base pool were purchased from the Institute of Liver Diseases of Beijing People's Hospital. They were HIV _{1 + 2} antigen, HBs antigen and HCV antigen, and their spotting concentrations were between 1.0-1.5 mg / ml. A substrate probe area 0.5 mm away from the highly hydrophobic convex body in the substrate pool Here, the three kinds of antigens are fixed by a known probe spotting method. Each antigen spot was 3 spots to form a 3 X 3 probe array. The chips were blocked with bovine serum albumin and used.

The chip detection device used in this embodiment is a chip detection device containing a synchronous purification system prepared in Example 1.

The chip detection method used in this embodiment is as follows:

1) Sample preparation-In this example, sample 1 is HCV antibody-positive serum, sample 2 is HIV _{1 + 2} antibody-positive human serum, sample 3 is HBs antibody-positive human serum, and sample 4 is a negative control . All samples were determined in advance using the classic single-reactor open chip under the same reaction conditions. All samples were diluted 20-fold with PBS buffer for testing.

2) Add sample:

Use a sample gun to add samples, and add 10 μl samples to each reaction cell.

3) Response

The reaction temperature was 37 degrees and the time was 15 minutes.

4) Removal and washing of reaction residues:

After the reaction is completed, the chip is rotated by 180 degrees (with the reaction cell facing downward), and then transported to the isolation purification chamber of the multi-reactor synchronous purification system via a transport belt, and then the fluid is cleaned.

When using a serial dilution purification system, the flow rate of the PBS wash solution flowing in and out of each reaction cell is 2 ml / min. Purification time: 1 minute.

When using a pressure fluid synchronous purification system, the working parameters are as follows: A. Use 1 # multiple nozzles (see Table 1), and each outlet is aligned with the probe array in a reaction cell; B. Use an angle adjuster to make the pressure fluid outlet The clockwise angle between the direction of liquid flow and the base plane of the fixed probe of the chip is 90 ± 5 degrees, even if the fluid direction is from low to high (spray upward); C. Use a torque regulator to make the pressure fluid outlet and The distance between the substrate planes is 2.0 ± 0.3 cm (the chip thickness in this embodiment is 1.0 ± 0.1 mm); D. the working hydraulic pressure of the pressure fluid at the outlet is 2.5 ± 0.5 kg / cm ² with a pressure regulator. Purification time is 0.5 minutes.

When using a pressure / ultrasonic fluid synchronous purification system, the operating parameters are the same as when using a pressure fluid synchronous purification system. But ultrasonic waves occur between the pressure pump and the nozzle outlet The power of the device is adjustable from 10 to 100 watts. Purification time: 0.5 minutes

5) Marker reaction

Rotate the chip 180 degrees (with the reaction cell facing upwards), and blow dry with air at 37-39 ° C, and then add 10 μl of labeling liquid to each reaction cell. The marker used was rhodamine-labeled goat anti-human secondary antibody. The reaction temperature was 37 ° C and the reaction time was 20 minutes.

6) Removal and washing of marker residues

After the reaction is completed, the chip is rotated 180 degrees (with the reaction cell facing downward), and then the above-mentioned multi-reactor synchronous purification system is used to remove and wash the marker residues. After washing, rotate the chip 180 degrees (with the reaction cell facing up), and blow dry the chip with air at 37-39 ° C.

7) Reading and analysis of detection light signal

The blown chip is transferred to a confocal laser scanner (Afymetrix GMS 418) via a conveyor belt. The scanning parameters are: the scanning excitation light wavelength is 532 nm, the emission light wavelength is 570 nm, and the laser intensity and gain are 60/69 respectively. The read signal is processed by the processing software JAGUAR II, and then the average value is obtained. No cross-contamination was observed with different multi-reactor simultaneous purification systems, and the negative and positive results on each reaction cell were consistent with the samples used.

Compared with the existing purification methods, the purification method of the present invention has the advantages of shorter purification time and high repeatability. In particular, according to the purifying method of the present invention by flushing with pressure fluid, no target signal loss has been observed in experiments where the working hydraulic pressure at the outlet exceeds 5.0 kg / cm ² , the background of the washed chip is clean, and the occurrence rate of the marks on the chip background is similar to The existing purification method is more than doubled. It is well known that low background noise is conducive to improving sensitivity. Example 6: Chip detection method with thickening and simultaneous purification

In this embodiment, the thickening means increasing the viscosity of the reaction residue by a specified amount; the simultaneous purification method means removing and washing the reaction residue at the same time.

The chip, sample, and marker used in this embodiment are the same as those used in Example 5. The chip detection device used is the system containing the thickening system and the simultaneous purification system in Example 2. Integrated chip detection device.

The chip detection method used in this example is the same as the chip detection method used in Example 5, except that after the sample addition reaction, before the reaction residues are removed and washed, a thickening step is added: When the component thickening system is used, The thickener is dry glucose powder. 10-15 mg of dry glucose powder is added to each reaction cell, so that the reaction residue no longer exists in liquid form but in the form of adsorbed water.

When using a physical thickening system, the temperature is set to 37 ° C, the reaction time is set to 15 minutes, and the humidity is set to a preferred humidity (relative humidity 55%) such that the volume reduction rate is less than 20%.

No cross-contamination was observed with different viscosifying systems, and the negative and positive results obtained on each reaction cell were consistent with the samples used. Example 7: Chip detection method with thickening, simultaneous purification and background signal attenuation

In this embodiment, the thickening and synchronous purification are the same as those in Embodiment 6. The weakening of the background signal means that the background signal is attenuated by a high absorbance structure outside the chip.

The chip, sample, and marker used in this embodiment are the same as those used in Example 6. The chip detection device used is the chip detection device prepared in Example 3, which includes a thickening system, a synchronous purification system, and a background signal attenuation system.

The chip detection method used in this embodiment is the same as the chip detection method used in Embodiment 6, except that the reading of the detection optical signal is performed in a scanner with a high absorbance structure.

In a scanner without a high absorbance structure, the background signal reading of the chip in this embodiment is between 50 and 1000; and in a scanner with a high absorbance structure, the background signal reading of the chip is 10 in this embodiment. — Between 100. It is well known that low background signals are conducive to improving sensitivity. The negative and positive results obtained on each reaction cell are consistent with the samples used. Example 8: Chip detection method with thickening, simultaneous purification and background signal enhancement

In this embodiment, the thickening and synchronous purification are the same as those in Embodiment 6. The background signal enhancement refers to strengthening the background signal with a light-emitting or / and reflective structure outside the chip.

The chip, sample and marker used in this embodiment are the same as those used in Example 6. The chip detection device used is the viscosity-increasing system, synchronous purification system, background signal attenuation system, and background signal enhancement system prepared in Example 4. Chip detection device.

The chip detection method used in this embodiment is the same as the chip detection method used in Embodiment 6. Similarly, the reading of the detection light signal is performed in a scanner containing a light emitting structure. In a scanner without a high absorbance structure / light emitting structure, the background signal reading of the chip in this embodiment is between 50-1000; and in a scanner containing a light emitting structure, the chip background signal reading in this embodiment is between Between 10000 and 20000. Using the method of this embodiment, the negative and positive results obtained on each reaction cell are consistent with the sample used, and the detection sensitivity is improved (after the positive sample is diluted 100 times, the chip containing the viscosity-increasing, simultaneous purification and background signal enhancement of the present invention is used). The results obtained by the detection method are positive, while the results obtained by using the existing chip detection method without thickening, simultaneous purification and background signal enhancement are negative).

Claims

Claim

1. A multi-reactor chip detection method, comprising at least one of the following steps: A. Quantitatively increasing the viscosity of the reaction residue; B. Simultaneously removing and washing the reaction residue of the reactor; C. Taking Luminous or / and reflective structures not on the chip reinforce the background signal; and D. Attenuate the background signal with a high absorbance structure not on the chip; wherein the multi-reactor chip contains multiple reactions formed on the same substrate The multi-reactor chip detection method includes a detection method using the multi-reactor chip. The reaction residue refers to an unfixed sample and optionally existing after the immobilization reaction in the reactor is completed. In the reagent, the high absorbance structure is a structure having a signal light absorption rate greater than 95%, preferably greater than 97%.

2. The multi-reactor chip detection method according to claim 1, wherein the removal and washing of the reaction residues of the reactor are performed simultaneously under the action of a fluid.

3. The multi-reactor chip detection method according to claim 2, wherein the fluid comprises a fluid containing pressure energy.

4. The multi-chip detection method according to claim 3 reactor, wherein the fluid is a liquid, and the hydraulic pressure 0.1- 10.0 kg / cm ^2, preferably 1.5_5.0 kg / cm ^2, more preferably 2.0 to 4.0 kg / cm ² .

5. The multi-reactor chip detection method according to any one of claims 1 to 4, wherein the viscosity of the reaction residue is increased by 10% or more, preferably 20% or more.

6. The multi-reactor chip detection method according to any one of claims 1 to 5, wherein the background signal is strengthened to more than 200%, preferably more than 500% of a negative target signal, and the negative target signal is a negative sample. The obtained detection signal.

7. The multi-reactor chip detection method according to any one of claims 1 to 5, wherein the background signal is weakened to less than 80%, preferably less than 70% of a negative target signal.

8. A chip detection device, comprising at least one of the following functional systems: A. A thickening system for increasing the viscosity of the reaction residue; B. Synchronizing the removal and washing of the reaction residue from the reactor A purification system; C. a background signal enhancement system including the light-emitting or / and reflective structure; and D. a background signal attenuation system including the high absorbance structure.

9. The chip detection device according to claim 8, wherein the synchronous purification system Includes pump and sprinkler.

10. The chip detection device according to claim 9, wherein the nozzle has a plurality of outlets for the fluid, and the number and density of the outlets are not less than the number and density of reactors of the multi-reactor chip.

11. The chip detection device according to claim 10, wherein the synchronous purification system has one or more of the following operating parameters: A. The outlet density is greater than 0.5 / cm ² , preferably greater than 1 / cm ² ; B The clockwise angle between the direction of flow of the fluid at the outlet and the base plane of the fixed probe of the chip is between 5 and 350 degrees, preferably 90 ± 5 degrees or 180 ± 5 degrees; C. The distance between the outlet and the substrate plane of the reactor is between 0.1 and 10.0 cm, preferably between 1 and 3 cm; D. The working hydraulic pressure of the outlet is between 0.1 and 10.0 kg / cm ^2, preferably between 1.5 and 5.0. kg / cm ² , more preferably 2.0-4.0 kg / cm ² .

12. The chip detection device according to any one of claims 8 to 11, wherein the viscosity increasing system comprises a temperature controller or / and a humidity controller.

13. The chip detection device according to any one of claims 8 to 12, wherein the thickening system contains a thickener transporting device.

14. The chip detection device according to any one of claims 8 to 13, wherein the background signal enhancement system includes parts and components of a coating containing a substance capable of emitting a light signal.

15. The chip detection device according to any one of claims 8 to 13, wherein the background signal attenuation system includes parts and components including a coating having a detection light reflectance of less than 3%.

16. The chip detection device according to claim 14 or 15, wherein the surface roughness Ra of the coating is between 0.2 and 3 μm, preferably between 0.5 and 2 μm.