Patent Description:
Biochip technology is a high-throughput biomolecular detection technology integrating microelectronics, life sciences, computer science and photo-electrochemistry, and is a major revolution in the field of life sciences. The traditional form of biochip technology is also called microarray technology. Its principle is to integrate biomolecules (DNA, RNA, peptides, proteins, etc.) with known sequences on a solid surface to form a probe array. Labeled biomolecules to be detected are used for hybridization reaction with the above probe array, and the purpose of biomolecule detection is achieved by detecting the hybridization probe at the corresponding position. The traditional biochip hybridization belongs to solid-liquid phase hybridization, and its discrete solid-liquid reaction environment and washing factors shows some shortcomings in detection sensitivity and detection of rare samples.

Suspended liquid biochip is a new high-throughput multiple detection technology that integrates molecular biology, immunology, polymer chemistry, optical detection technology, microfluidic technology, and computer technology based on SAT (Suspension Array Technology). This technology well overcomes the technical shortcomings of solid state array chips, and has advantages such as high throughput, multiple indicators, high sensitivity (~ <NUM> pg/mL), high specificity, wide linear range (up to <NUM> - <NUM> orders of magnitude), fast response (<NUM> - <NUM>), good repeatability and easy operation and so on compared with other detection methods. Therefore, it is currently the only biochip technology and product approved by the US Food and Drug Administration (FDA) for clinical diagnosis. Its application in clinical diagnosis have involved immunological analysis, DNA hybridization analysis, and molecule detection of protein and gene expression profile and other fields. Biochip technology is considered to be the key development direction of clinical medical testing, so it has been included in National Science and Technology Innovation Plan of China's "13th Five-Year" for in vitro diagnostic technologies that require breakthroughs.

<CIT> discloses a suspended biochip comprises a polymer microsphere as a carrier, a suspension medium for maintaining the microsphere in a suspended state, a biological probe and a container for holding the biochip. The microspheres coated with probes are mixed in a certain proportion in the suspension medium, and microspheres of different sizes are used as carriers for different biological probes. The size of the microsphere corresponds one to one with the type of probe.

<NPL>) discloses different methods of preparing encoded particles, in which optical encoding methods comprising optical encoding with organic dyes and optical encoding with photoluminescent nanoparticles are disclosed.

<NPL>) discloses a combined membrane emulsification-solvent evaporation (MESE) approach for the efficient preparation of QD barcodes. By combining the advantages of the MESE approach in controlling the barcode sizes with accurate encoding, a three dimensional barcode library that integrates the signals of the forward scattering, fluorescence <NUM>, and fluorescence <NUM> channels was established via flow cytometry. The five indexes of hepatitis B viruses were chosen as diagnostic targets to examine the feasibility of the QD barcodes in high-throughput diagnosis. The ability of these QD barcodes to simultaneously and selectively detect a multitude of diverse biomolecular targets was also demonstrated.

<CIT> discloses a test tube holder which will accommodate test tubes of various sizes, a test tube holder which is adjustable in height, a test tube holder which permits of ready view of the contents of the tubes, and a test tube holder which is sturdy, exceedingly useful in operation and inexpensive to manufacture.

<CIT>, <CIT> and <CIT> relate to various tube/cuvette holder arrangements.

At present, the existing commercial liquid biochip products still have technical problems to be solved: <NUM>) the fluorescent dyes used in the existing liquid biochip microspheres on the market are mostly organic dyes and when multicolor labeling is performed, the wide emission peaks make the signals easy to overlap and difficult to distinguish; <NUM>) the poor light stability of organic fluorescent dyes and the poor ability of organic fluorescent dyes to resist fluorescent bleaching would affect their service life or storage period; <NUM>) the classification fluorescence on the microsphere probe and the report fluorescence of the molecule to be test in the sample must be excited by laser devices with two different wavelengths, which leads to a higher cost of the detection instrument; <NUM>) this type of detection instrument has only two detection channels, and also has strict requirements on the size of the microspheres (around <NUM>), thus the fluorescence signal parameter setting is greatly restricted, which is not conducive to the future needs and applications for high-throughput and rapid clinical testing.

At present, the conventional method using suspension liquid biochip has a relatively expensive system and a high cost for a single test sample, and difficult for application. Therefore, there is an urgent need for a low-cost and easy-to-operate detection technology using suspension liquid biochip in practical applications.

One of the objectives of the present invention is to overcome the deficiencies in the prior art. There is provided a detection system using suspension liquid biochip as defined with appended independent claim <NUM>. Preferred embodiments of the invention are defined with the dependend claims.

As additional information and outside the scope of the invention as defined with the claims, a detection method using suspension liquid biochip and a detection system using suspension liquid biochip is described.

The detection method using suspension liquid biochip comprises the steps:.

The wavelength of the laser light emitted by the single laser device is <NUM>-<NUM>.

Lasers of the same wavelength are used to simultaneously excite the classification fluorescence of the nanocrystalline fluorescent microspheres and the report fluorescence of the object to be detected.

The classification fluorescence is fluorescence of at least one or more wavelengths.

The classification fluorescence includes fluorescence of more than two wavelengths.

The report fluorescence of the object to be detected is realized via the excitation of a fluorescent dye by the laser; the fluorescent dye is one or more of fluorescein isothiocyanate, phyllochlorin, phycoerythrin, anthocyanin, or surface-modified semiconductor quantum dots. Fluorescein isothiocyanate is abbreviated as FITC; phyllochlorin is abbreviated as PerCP; phycoerythrin is abbreviated as PE; anthocyanin is abbreviated as CY5.

In the signal acquisition step, the acquisition channel includes one or more of FSC, FL1, FL2 and FL3.

Two of the acquisition channels are used to acquire the signals of the classification fluorescence.

One of the acquisition channels is used to acquire the signal of the report fluorescence.

After the signal acquisition step, a result calculation step is further included, and the following steps are sequentially included in the result calculation step: a. the signals acquired by the FSC channel are made into a histogram, and the data in a selected range near a peak value are selected, and the signal data related to the fluorescent microspheres within the selected data range are used in the next calculation; b. the acquired signals of the classification fluorescence corresponding to the nanocrystalline fluorescent microspheres selected in step a are used to make a classification fluorescence scatter diagram; c. an effective area for the scatter diagram of each nanocrystalline fluorescent microsphere is selected, and the corresponding data in the effective area are selected; d. a report fluorescence signal distribution histogram of the nanocrystalline fluorescent microspheres in the effective area selected in step c is made and a peak value data corresponding to the report fluorescence signals acquired from these nanocrystalline fluorescent microspheres is selected, and a median value of the result data is calculated; e. a concentration result corresponding to an intensity of the report fluorescence signals is obtained through a standard curve.

The classification fluorescence signals of each type of nanocrystalline fluorescent microsphere are pre-coded before the detection in the step b.

In the step d, for particle cluster of each type of nanocrystalline fluorescent microsphere in the effective area, the peak value data of the report fluorescence signal is calculated by the area under the signal curve, the median value of the result data is calculated, and the intensity of the report fluorescence signal is obtained.

According to the invention, a detection system using suspension type liquid biochip is provided as defined with claim <NUM>, comprising a housing; the housing encloses a chamber; a clamp is installed on the housing, and the clamp is used to clamp a tube; the clamp is rotatably arranged on the housing, and the clamp is provided with a first clamping hole and a second clamping hole; the first clamping hole is used to fix a first tube, and the second clamping hole is used to clamp a second tube; the axis of the first clamping hole intersects the axis of the second clamping hole; the clamp is rotatably arranged, and when the clamp rotates, the opening of the first tube or the opening of the second tube faces a direction suitable for use.

According to an embodiment of the present invention, the axis of the first clamping hole is perpendicular to the axis of the second clamping hole.

According to an embodiment of the present invention, the clamp includes a base and a plurality of clamping jaws, and the plurality of clamping jaws are arranged in two horizontal columns and arranged in two vertical rows on the base; the two horizontal columns are arranged at intervals, and the first clamping hole is located between the two horizontal columns; the two vertical rows are arranged at intervals, and the second clamping hole is arranged between the two vertical rows.

According to an embodiment of the present invention, the number of the clamping jaws is four, and the four clamping jaws are arranged on the base at intervals according to a rectangular distribution; the four clamping jaws form two vertical rows and two horizontal columns; the two vertical rows of clamping jaws are used to clamp the first tube; the two horizontal columns of clamping jaws are used to clamp the second tube.

According to an embodiment of the present invention, the base is rotatably mounted on the housing.

According to an embodiment of the present invention, the base comprises a connecting post, a plurality of catch grooves are provided on the surface of the connecting post; the plurality of catch grooves are distributed along the circumferential direction; the connecting post is rotatably installed on the housing;
the housing is further provided with a restricting member and the restricting member is movably arranged;.

When the restricting member moves, the end of the restricting member can be inserted into the catch groove and can be configured to be able to withdraw from the catch groove; when the end of the restricting member is inserted into the catch groove, the rotation of the connecting post is restricted; after the end of the restricting member withdraws from the catch groove, the restriction on the connecting post is released;
when the connecting post rotates against the restricting member, the restricting member can move to make the end exit the catch groove and when the next catch groove rotates to be opposite to the end of the restricting member, the end of the restricting member is inserted into the catch groove.

According to an embodiment of the present invention, it further includes an elastic reset device, the elastic reset device is configured to deform when the restricting member moves; the elastic reset device is used to reset the restricting member.

According to an embodiment of the present invention, the elastic reset device is pre-deformed to generate an elastic force, the elastic force keeps the end of the restricting member in a state of being inserted into the catch groove; when the restricting member moves, the deformation of the spring is greater.

According to an embodiment of the present invention, the restricting member is provided with a recess, and the elastic reset device is a cylindrical spring; one end of the cylindrical spring abuts against the recess, and the other end abuts against the housing.

According to an embodiment of the present invention, a mounting plate is arranged in the recess, the mounting plate is provided with a positioning post, and the end of the cylindrical spring is sleeved on the positioning post and abuts against the mounting plate.

According to an embodiment of the present invention, the positioning post is cross-shaped, and the positioning post is further provided with a convex column, the convex column is inserted into the cylindrical spring and thus connects the cylindrical spring with the positioning post.

According to an embodiment of the present invention, the housing is provided with a transparent plate through which the chamber can be observed.

According to an embodiment of the present invention, the housing is provided with an openable and closable door plate; the door plate is opposite to the cleaning liquid bottle provided in the chamber, and the cleaning liquid bottle can be taken out or put in after opening the door plate; the transparent plate is arranged on the door plate.

The classification fluorescence is the fluorescence emitted by the nanocrystalline fluorescent microspheres after being excited by laser, and is used for encoding to distinguish different nanocrystalline fluorescent microspheres. Report fluorescence is fluorescence used to mark a object to be tested for the detecting instrument to detect the presence of the object to be tested and the content of the object to be tested.

The optical path system is provided with a single laser device.

Compared with the prior art, the following beneficial effects are mentioned:.

As shown in <FIG>, a detection system <NUM> using suspension liquid biochip includes a liquid path system, an optical path system, a detection and analysis system, and a housing <NUM>. The liquid path system includes structures such as a sheath liquid barrel <NUM> and a waste liquid barrel <NUM>. The liquid path system can use the structure in the prior art, which would not be repeated here. In the present invention, the optical path system can adopt the corresponding structures in the prior art except that only one laser device is provided. The optical path system in the present invention is provided with a single laser device (not shown in the figure). The single laser device is used to emit a laser of a selected wavelength, for example, a <NUM> laser light. The specific laser wavelength range can be determined according to actual needs. The detection and analysis system can also adopt the corresponding structure in the prior art.

As shown in <FIG>, the housing <NUM> is composed of an upper cover <NUM>, a front door <NUM>, two side wall plates <NUM>, a bottom plate <NUM> and a rear wall plate (not shown in the figure). The housing <NUM> encloses a cavity <NUM>. A recess <NUM> is provided on one corner of the housing <NUM>. A mounting hole (not shown in the figure) is provided in the recess <NUM>. The mounting hole is used for mounting a clamp <NUM> described later.

As shown in <FIG>, the upper cover <NUM> is configured to be openable. After the upper cover <NUM> is opened, detection can be performed. As shown in <FIG>, <FIG>, one of the side wall plates <NUM> is provided with an openable and closable door plate <NUM>; the door plate <NUM> is opposite to the cleaning liquid bottle <NUM> provided in the chamber <NUM>, and the cleaning liquid bottle can be taken out or put in after opening the door plate <NUM>. The door plate <NUM> is provided with a transparent plate <NUM> through which the cleaning liquid in the cleaning liquid bottle inside the chamber <NUM> can be observed. The transparent plate <NUM> has a strip shape extending vertically. As shown in <FIG>, the front door <NUM> is configured to be openable.

As shown in <FIG>, a restricting member <NUM> is provided in the chamber <NUM>. The restricting member <NUM> has a substantially cuboid shape. The restricting member <NUM> has one end <NUM>. The restricting member <NUM> is also provided with a recess <NUM>. The recess <NUM> is used for accommodating a part of the cylindrical spring <NUM>, which can ensure that the cylindrical spring <NUM> is installed more stably and more securely during use. A mounting plate <NUM> is provided in the recess <NUM>. A positioning post <NUM> is provided on the mounting plate <NUM>. The positioning post <NUM> is cross-shaped. A convex column <NUM> is also provided on the positioning post <NUM>. The restricting member <NUM> is configured to be movable up and down. The restricting member <NUM> is connected with a cylindrical spring <NUM>. One end of the cylindrical spring <NUM> is sleeved on the positioning post <NUM> and abuts against the mounting plate <NUM>. The convex column <NUM> is inserted into the cylindrical spring <NUM> to clamp the cylindrical spring <NUM>, so that the connection between the positioning post <NUM> and the cylindrical spring <NUM> is more stable. The other end of the cylindrical spring <NUM> is abutted against the inside of the housing <NUM>. When the restricting member <NUM> moves downward, the cylindrical spring <NUM> can generate elastic force due to deformation. The elastic force can make the restricting member <NUM> move upward and reset.

The inspection system <NUM> in the present invention further includes clamp <NUM>. The clamp <NUM> is used to clamp a tube. The clamp <NUM> is provided with a first clamping hole <NUM> and a second clamping hole <NUM>. The first clamping hole <NUM> is used for a first tube <NUM> inserting. The second clamping hole <NUM> is used for a second tube inserting (not shown in the figure). The axis of the first clamping hole <NUM> and the axis of the second clamping hole <NUM> intersect, and preferably the axes of the two intersect perpendicularly.

The clamp <NUM> includes a base <NUM> and four clamping jaws <NUM>. The base <NUM> includes a connecting post <NUM>, a bottom plate <NUM> and a connecting rod <NUM>. The bottom plate <NUM> and the connecting post <NUM> are connected by the connecting rod <NUM>. A catch groove <NUM> is provided on the surface of the connecting post <NUM>. There are a plurality of catch grooves <NUM> evenly distributed along the circumferential direction. In the example shown in the figure, four catch grooves <NUM> are provided, which are evenly distributed along the circumferential direction.

The four clamping jaws <NUM> are arranged on the bottom plate <NUM> in a rectangular distribution and at intervals, and protrude from the bottom plate <NUM>. The four clamping jaws <NUM> form two vertical rows and two horizontal columns. The gap between the two horizontal columns of clamping jaws <NUM> is a first clamping hole <NUM> for clamping the first tube <NUM>. The gap between the two vertical rows of clamping jaws <NUM> is a second clamping hole <NUM> for clamping the second tube (not shown in the figure).

The connecting post <NUM> of the clamp <NUM> is inserted in the mounting hole and can rotate. The connecting post <NUM> is located above the end <NUM> of the restricting member <NUM>. The end <NUM> of the restricting member <NUM> can be inserted into the catch groove <NUM> to limit the rotation of the connecting post <NUM>. The end <NUM> of the restricting member <NUM> can also be withdrawn from the catch groove <NUM> to release the restriction on the connecting post <NUM>. The cylindrical spring <NUM> is pre-compressed to generate elastic force, and the elastic force keeps the end <NUM> of the restricting member <NUM> in the catch groove <NUM>. When the clamp <NUM> needs to be rotated, the base <NUM> is rotated forcefully, and the connecting post <NUM> presses the end <NUM> of the restricting member <NUM> so that the end <NUM> exits the catch groove <NUM>. During the rotation of the connecting post <NUM>, until the other catch groove <NUM> rotates above the end <NUM> of the restricting member <NUM>, the elastic force of the cylindrical spring <NUM> causes the restricting member <NUM> to move upward and reset until the end <NUM> is inserted into the catch groove <NUM>, and The connecting post <NUM> is restricted to this state. The arc between the two catch groove <NUM> is <NUM> degrees, and the connecting post <NUM> is clamped every time it rotates <NUM> degrees. During the rotation of the clamp <NUM>, the opening of the first tube <NUM> clamped by the clamp <NUM> or the opening of the second tube clamped by the clamp <NUM> may face a direction suitable for use, such as upward. The connecting post <NUM> is provided with a catch groove <NUM> to cooperate with the end <NUM> of the restricting member <NUM>. When different tubes are installed, it only needs to be rotated to make the opening of the tube suitable for operation, which is convenient to use. The cylindrical spring <NUM> helps to stably restrict the connecting post <NUM>, which can prevent connecting post <NUM> from loosening and allow connecting post <NUM> to rotate. Therefore, the clamp <NUM> is provided with the first clamping hole <NUM> and the second clamping hole <NUM>, which can clamp a test tube or a centrifuge tube and other different tubes, which is more convenient to use.

The detection method using suspension liquid biochip uses the detection system <NUM> using suspension liquid biochip in Example <NUM>, and includes the following steps:.

In this example, two types of nanocrystalline fluorescent microspheres are used, both of which are coded by fluorescence; the surface of one type of the nanocrystalline fluorescent microspheres is coupled to the anti-alpha-fetoprotein antibody AFP-Ab; the other type of the nanocrystalline fluorescent microspheres is coupled to the anti-carcinoembryonic antigen antibody CEA-Ab, which respectively form two biological detection probes for specific detection of alpha-fetoprotein AFP and specific detection of carcinoembryonic antigen CEA. These two biological detection probes are added to the serum sample to react.

Finally, the target connected to the biological detection probe is labeled with two phycoerythrin fluorescein which are respectively coupled with specific antibodies against the AFP and CEA antigens. Here, the specific antibodies respectively coupled with AFP and CEA antigens are used as secondary antibodies. The fluorescence emitted by the phycoerythrin fluorescein excited by the laser is the report fluorescence of the object to be detected. Phycoerythrin fluorescein emits fluorescence with a wavelength of <NUM> after being excited by laser.

Step <NUM>. Excitation step: the nanocrystalline fluorescent microspheres obtained in step <NUM> are excited by a single laser device, and the fluorescence signal is detected by a detecting instrument. The detecting instrument is a single-light three-color detecting instrument, i.e. the detecting instrument has a single laser device and collects fluorescence signals of three wavelengths. Moreover, in this example, the single laser device emits laser light of the same wavelength to excite classification fluorescence and report fluorescence, i.e. three different wavelengths of fluorescence are excited. In this example, the wavelength of the laser light emitted by the laser device is <NUM>.

Step <NUM>: Signal acquisition step: classification fluorescence and report fluorescence detection signals are obtained through multiple acquisition channels, wherein the acquisition channels include one or more of FSC, FL1, FL2, and FL3. The selected specific number can be determined according to the number of fluorescence wavelengths. In addition to the FSC channel, at least two of the acquisition channels are used to acquire the classification fluorescence signals of the nanocrystalline fluorescent microspheres. In this example, the FSC channel acquires fluorescence signals related to the particle size of the nanocrystalline fluorescent microspheres, and the other two channels (FL1 and FL2) acquire fluorescence signals around <NUM> and <NUM>, respectively. <NUM> and <NUM> fluorescence are classification fluorescence and used to detect different nanocrystalline fluorescent microspheres. The acquisition channel FL3 is used to acquire the signal of the report fluorescence, i.e. to acquire the fluorescence signal near <NUM>.

Step <NUM>. Result calculation steps: the result calculation process is shown in <FIG>, including the following steps:.

In the above example, only two wavelengths of fluorescence are combined in different proportions for encoding. According to the technical solution of the present invention, the particle size of the nanocrystalline fluorescent microspheres can also be used for encoding. For example, a particle size of <NUM>, a particle size of <NUM>, and a particle size of <NUM>, plus two or more wavelengths of fluorescence. In this way, more codes can be realized to distinguish more microspheres, and more targets can be detected at the same time in the same batch.

Claim 1:
A detection system (<NUM>) using suspension liquid biochip, comprising a housing (<NUM>); the housing encloses a chamber (<NUM>); a clamp (<NUM>) is installed on the housing, and the clamp is used to clamp a tube; characterized in that the clamp is rotatably arranged on the housing, and the clamp is provided with a first clamping hole (<NUM>) and a second clamping hole (<NUM>); the first clamping hole is used for a first tube (<NUM>) inserting, and the second clamping hole is used for a second, different tube inserting; an axis of the first clamping hole intersects an axis of the second clamping hole; the clamp is rotatably arranged, and when the clamp rotates, an opening of the first tube or an opening of the second tube faces a direction suitable for use.