CN113702338A - Multichannel biological reaction sensing chip and manufacturing method and device thereof - Google Patents
Multichannel biological reaction sensing chip and manufacturing method and device thereof Download PDFInfo
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Abstract
The invention provides a multi-channel biological reaction sensing chip and a manufacturing method and a device thereof, wherein the manufacturing method comprises the following steps: the probe comprises a sensing chip body and a plurality of probe channels arranged on the sensing chip body, wherein different types of probe molecules are respectively fixed on the probe channels. According to the invention, the plurality of probe channels fixed with different types of probe molecules are arranged on the sensing chip body, so that the specificity analysis and content detection of various target molecules in a biological sample can be realized, and the sensing efficiency is improved.
Description
Technical Field
The invention belongs to the technical field of biomacromolecule optical sensing, and particularly relates to a multichannel biological reaction sensing chip and a manufacturing method and device thereof.
Background
The Surface Plasmon Resonance Imaging (SPRi) sensing technology has the characteristics of high sensitivity, no labeling and high flux, can detect the interaction condition of biomacromolecules, is widely applied to the fields of biosensing, environmental monitoring and food safety, and especially plays an extremely important role in biomacromolecule combination (such as antigen-antibody and the like) and detection of kinetic parameters thereof.
Although the SPRi sensing technology can realize the binding of a biological probe and a chip and the specific binding detection of a target molecule in a biological sample and the biological probe, the traditional SPRi biological sensing chip has low sensing efficiency and a complex flow system, and is difficult to be used in the fields of clinical detection and the like.
Therefore, the prior art is subject to further improvement.
Disclosure of Invention
In view of the defects in the prior art, the invention aims to provide a multi-channel biological reaction sensing chip and a manufacturing method and a manufacturing device thereof, which overcome the defect of low sensing efficiency of the conventional SPRi biological sensing chip.
The first embodiment disclosed by the invention is a multi-channel biological reaction sensing chip, which comprises: the probe comprises a sensing chip body and a plurality of probe channels arranged on the sensing chip body, wherein different types of probe molecules are respectively fixed on the probe channels.
The multichannel biological reaction sensing chip comprises a sensing chip body and a substrate, wherein the sensing chip body comprises a glass substrate and a metal film arranged on the glass substrate.
The multi-channel biological reaction sensing chip is characterized in that the probe channels are arranged on the sensing chip body at equal intervals and are parallel to each other.
The second embodiment of the present invention is a method for manufacturing the multi-channel biological reaction sensor chip, which includes:
attaching a prefabricated mold to the surface of the sensing chip body; the mould comprises a plurality of concave parts, and when the mould is attached to the surface of the biochip body, a plurality of first microflow channels are formed between the concave parts and the biochip body;
respectively introducing a plurality of different types of probe molecule solutions into the plurality of first micro-flow channels, and enabling the plurality of different types of probe molecule solutions to be in contact with the surface of the biochip body through the plurality of first micro-flow channels to form a plurality of probe channels on the biochip body; the inner surfaces of the plurality of concave parts are in a sawtooth shape, and the flow mode of the probe molecule solution in the plurality of first microflow channels is a turbulent flow mode with non-uniform flow speed.
The manufacturing method of the multichannel biological reaction sensing chip comprises a plurality of first sample inlets and a plurality of first sample outlets, wherein the plurality of first sample inlets and the plurality of first sample outlets are in one-to-one correspondence with the plurality of first micro-flow channels, and each first micro-flow channel is communicated with the first sample inlet and the first sample outlet corresponding to the each first micro-flow channel.
The manufacturing method of the multi-channel biological reaction sensing chip comprises the following steps of respectively introducing a plurality of different types of probe molecule solutions into a plurality of first micro-flow channels:
and adjusting the pressure difference between the first sample inlet and the first sample outlet corresponding to each first micro-flow channel, and controlling the probe molecule solutions of different types to flow back and forth in the first micro-flow channels by the pressure difference.
The third embodiment disclosed by the invention is a multi-channel biological reaction sensing device, which comprises the multi-channel biological reaction sensing chip.
The multi-channel biological reaction sensing device, wherein, the device also includes: and each sample channel unit is orthogonally attached to the probe channels.
The multichannel biological reaction sensing device is characterized in that each sample channel unit comprises a second sample inlet, a plurality of second microfluidic channels and a second sample outlet, and the second sample inlet and the second sample outlet are communicated with the plurality of second microfluidic channels; after the biological sample is introduced into the sample channel unit from the second sample inlet, the biological sample repeatedly flows in the second microfluidic channels by changing the pressure difference between the second sample inlet and the second sample outlet.
The multichannel biological reaction sensing device is characterized in that a sawtooth-shaped channel structure is arranged on the side wall of each second microfluidic channel.
The multichannel biological reaction sensing chip has the beneficial effects that the plurality of probe channels fixed with different types of probe molecules are arranged on the sensing chip body, so that the specificity analysis and the content detection can be simultaneously carried out on various target molecules in a biological sample through the plurality of probe channels, and the sensing efficiency is improved.
Drawings
FIG. 1 is a schematic structural diagram of a multi-channel biological reaction sensor chip provided by an embodiment of the present invention;
fig. 2 is a schematic perspective view illustrating a die attached to a sensor chip body according to an embodiment of the present invention;
FIG. 3 is a side view of a die attached to a sensor chip body according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of a method for manufacturing a multi-channel bioreaction sensor chip according to an embodiment of the present invention;
FIG. 5 is a top view of a multi-channel bioreaction sensing device according to an embodiment of the present invention;
FIG. 6 is a schematic perspective view of a multi-channel biological reaction sensor device provided in an embodiment of the present invention;
FIG. 7 is a multi-channel signal graph of a multi-channel bioreaction sensing device according to an embodiment of the present invention;
FIG. 8 is a graph of the signal curve of the multichannel signal curve of FIG. 7 after normalization;
FIG. 9 is a schematic diagram of SPR responses at different measurement sites of the multi-channel biological reaction sensing device provided by the embodiment of the invention;
fig. 10 is a schematic structural diagram of a zigzag channel structure provided in an embodiment of the present invention.
The various symbols in the drawings: 1. a sensing chip body; 2. a probe channel; 3. a recessed portion; 4. a first microfluidic channel; 5. a probe molecule; 6. a first sample inlet; 7. a first sample outlet; 8. a sample channel unit; 81. a second microfluidic channel; 82. a second sample inlet; 83. and a second sample outlet.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the embodiments and claims, the terms "a" and "an" can mean "one or more" unless the article is specifically limited.
In addition, if there is a description of "first", "second", etc. in an embodiment of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
Surface Plasmon Resonance Imaging (SPRi) is a sensing technique for detecting changes in the Surface properties of biochips by optical methods. Generally, biochips are made by evaporating a thin film of a noble metal (e.g., gold, etc.) on a glass substrate. When a beam of light is incident on the surface of the chip, free electrons in the metal film are excited to generate plasma waves, the light waves and the plasma waves are coupled with each other, so that the physical properties (such as spectrum, intensity, phase and the like) of reflected light are changed along with the change of the surface property of the biochip, and the change of the physical properties of the reflected light is detected by a detector (CCD, spectrometer and the like) to represent the process of biological reaction on the surface of the chip, thereby achieving the purpose of sensing.
Although the SPRi sensing technology can realize the binding of a biological probe and a chip and the specific combination detection of target molecules and biological probes in a biological sample, the traditional SPRi biological sensing chip can only realize the detection of one target molecule by a single channel, and the device structure is mostly a one-way pipeline structure of sample introduction, a micro-flow cavity and sample discharge, bubbles are easily generated in the experimental process to block the micro-flow cavity and introduce experimental errors, in addition, the pipeline is easy to pollute, the pipeline needs to be replaced in time to avoid the introduction of pollution, the pipeline operation is complex, the efficiency is low, and the device is difficult to be used in the fields of clinical detection and the like.
In order to solve the above problems, the present invention provides a multi-channel biological reaction sensor chip, as shown in fig. 1, comprising: the sensor chip comprises a sensing chip body 1 and a plurality of probe channels 2 arranged on the sensing chip body 1, wherein different types of probe molecules are respectively fixed on the probe channels 2. For example, the multichannel biological reaction sensing chip comprises a sensing chip body 1, and a probe channel a, a probe channel B and a probe channel C which are arranged on the sensing chip body 1, wherein the probe channel a fixes a probe molecule a, the probe channel B fixes a probe molecule B, the probe channel C fixes a probe molecule C, and so on, the number of the probe channels 2 and the types of the probe molecules are increased, so that a plurality of probe molecules can be fixed on one multichannel biological reaction sensing chip, the specificity analysis and the content detection of a plurality of target molecules in a biological sample are realized, and the sensing efficiency is improved.
In a specific embodiment, the sensing chip body 1 includes a glass substrate and a metal thin film disposed on the glass substrate, the metal thin film is a noble metal thin film, and in a process of modifying probe molecules on the surface of the sensing chip body 1, the probe molecules free in a probe molecule solution are specifically bound on the surface of the metal thin film through covalent bonds or physical adsorption and the like. In one embodiment, the metal thin film is a gold film, and in the actual SPRi sensing process, a sensing chip is generally formed by using a chemically stable gold film and probe molecules fixed on the surface of the gold film, plasma resonance is generated by the gold film and polarized interference light, and the refractive index of the surface of the gold film is changed by the combination of the probe molecules on the gold film and target molecules in the biological sample, so that the physical properties such as phase, wavelength, intensity, etc. of the reflected light are changed to perform SPR detection on the target molecules in the biological sample.
In a specific embodiment, the plurality of probe channels 2 are disposed at equal intervals on the sensor chip body 1, and the plurality of probe channels 2 are parallel to each other. For example, the plurality of probe channels 2 include a probe channel a, a probe channel B, and a probe channel C, which are sequentially arranged, the interval between the probe channel a and the probe channel B is equal to the interval between the probe channel B and the probe channel C, and the probe channel a, the probe channel B, and the probe channel C are parallel to each other.
Based on the multi-channel biological reaction sensing chip, the invention also provides a manufacturing method of the multi-channel biological reaction sensing chip, which comprises the following steps:
s1, fitting the prefabricated mold on the surface of the sensing chip body; the mould comprises a plurality of concave parts, and when the mould is attached to the surface of the biochip body, a plurality of first microflow channels are formed between the concave parts and the biochip body;
s2, respectively introducing a plurality of different types of probe molecule solutions into the first micro-flow channels, and enabling the different types of probe molecule solutions to be in contact with the surface of the biochip body through the first micro-flow channels to form a plurality of probe channels on the biochip body.
In order to prepare the multi-channel biological reaction sensor chip, in this embodiment, a soft mold is manufactured in advance, as shown in fig. 2 and fig. 3, the soft mold includes a plurality of concave portions 3, then the prefabricated soft mold is tightly attached to the surface of the sensor chip body 1, and when the soft mold is attached to the surface of the sensor chip body 1, a plurality of first microfluidic channels 4 are formed between the plurality of concave portions 3 and the sensor chip body 1. Then, a plurality of different types of probe molecule solutions are respectively introduced into the plurality of first microfluidic channels 4, as shown in fig. 3, the different types of probe molecule solutions flow back and forth in the direction of the arrow in each first microfluidic channel 4, so that the probe molecules 5 free in the probe molecule solutions are bonded to the surface of the sensor chip body 1 in the ways of covalent bonds or physical adsorption, and the like, thereby realizing the binding of the different types of probe molecules 5, and forming a plurality of probe channels 2 on the surface of the sensor chip body 1. In one embodiment, the inner surfaces of the recesses 3 are zigzag, and the probe molecule solution flows in the first microfluidic channels 4 in a turbulent manner with a non-uniform flow rate
In a specific embodiment, with continuing reference to fig. 2, the mold further includes a plurality of first sample inlets 6 and a plurality of first sample outlets 7, the plurality of first sample inlets 6 and the plurality of first sample outlets 7 correspond to the plurality of first microfluidic channels 4 one-to-one, and each first microfluidic channel 4 is communicated with the first sample inlet 6 and the first sample outlet 7 corresponding to each first microfluidic channel 4. In the manufacturing process of the specific multi-channel biological reaction sensing chip, after a prefabricated mold is attached to the surface of the sensing chip body 1, a plurality of probe molecule solutions of different types are introduced into the plurality of first micro-flow channels 4 from the plurality of first sample inlets 6.
In one embodiment, the step of introducing the plurality of different types of probe molecule solutions into the plurality of first microfluidic channels in step S2 includes:
m1, adjust the pressure differential between the first sample inlet that each first miniflow passageway corresponds and the first appearance mouth, through pressure differential control a plurality of different kinds of probe molecule solution is in flow back and forth in a plurality of first miniflow passageways.
The experimental error that the biological pollution that no pipeline's design can avoid pipeline structure to arouse and lead to, this embodiment is bound in order to realize the high efficiency of no pipeline trace probe molecule, and it is leading-in with a plurality of different kinds of probe molecule solution a plurality of first miniflow passageway 4 back, adjust the pressure differential between the first introduction port 6 that adjusts each first miniflow passageway 4 and the first appearance mouth 7 that corresponds, through pressure differential control a plurality of different kinds of probe molecule solution are in flow back and forth in a plurality of first miniflow passageway 4 to realize that the high efficiency of no pipeline trace probe molecule is bound. For example, as shown in fig. 4, the pressure of the first sample inlet 6 corresponding to the first microfluidic channel is P1, the pressure of the first sample outlet 7 corresponding to the first microfluidic channel is P2, and when P1 is different from P2, the liquid level of the probe molecule solution in the first microfluidic channel is different, so that the probe molecule solution can be controlled to flow back and forth in the microfluidic cavity by controlling the surface pressure difference between the first sample inlet 6 and the first sample outlet 7, and efficient binding of the pipeless trace sample bioprobe is achieved.
Based on the multi-channel biological reaction sensing chip for SPR sensing, the invention also provides a multi-channel biological reaction sensing device, as shown in FIGS. 5 and 6, the multi-channel biological reaction sensing device comprises the multi-channel biological reaction sensing chip for SPR sensing and a plurality of sample channel units 8 arranged on the multi-channel biological reaction sensing chip, and each sample channel unit 8 is orthogonally attached to the plurality of probe channels 2 to form a molecular reaction pool. In the specific SPRi sensing process, different biological samples are introduced into each sample channel, so that specific analysis and content detection of various biological macromolecules can be realized. For example, the plurality of sample channel units 8 include a sample channel unit D, a sample channel unit E, and a sample channel unit F, and a biological sample D, a biological sample E, and a biological sample F can be respectively introduced into the sample channel unit D, the sample channel unit E, and the sample channel unit F, so as to implement the specific analysis and the content detection for three biological samples.
The method is influenced by the unevenness of the surface of the multi-channel biological reaction sensing chip and different binding conditions of probes at different positions, only one detection site can be formed under the condition that a single biological sample channel is orthogonally combined with the probe channel 2, the data acquisition range is limited, and the requirement on the preparation consistency of the chip is extremely high. In order to solve the above problems, as shown in fig. 5 and fig. 6, each sample channel unit 8 in this embodiment includes a plurality of second microfluidic channels 81, and after a biological sample is introduced into the sample channel unit 8, a plurality of channels can be simultaneously and simultaneously combined with probes, so that the biological sample is combined with probe molecules at different positions on the same probe channel 2, thereby enlarging the data acquisition range, eliminating experimental errors caused by inconsistent SPR signal responses at different positions of a multi-channel biological reaction sensor chip, improving the consistency of biological sample detection, reducing the preparation quality requirement and the preparation cost for the sensor chip, and facilitating industrial application. For example, as shown in FIG. 6, when the sample 1 contains only the target molecule specific to the probe A, SPR signals at the sites (first, second, third, fourth, fifth, sixth, etc.) in contact with the biological probe are at high values, and SPR signals at the remaining (fourth-ninth) sites are at low values; similarly, if only the probe B specific target molecule is contained, the SPR signal of the site (c), (c) and (c) is in a high value, and the SPR signal of the other sites (c), (B) and (c) is in a low value.
In one embodiment, when the number of the second microfluidic channels 81 is n, the n second microfluidic channels 81 and one probe channel 2 form n detection points, and the signal curves of the n detection points are respectively set as R1，R2，R3……Rn，RbNormalizing the obtained n signal curves for the background noise of the probe channel, wherein the signal curve obtained after normalization treatment is
As shown in fig. 9, since SPR signal response is sensitive to the interaction of specific binding between the sensing surface biological probe and the target molecule, if the two are specifically bound, several SPR signals are normalized to obtain a signal curve a, and as can be seen from the signal curve a, the signal response tends to be flat after rising; if the sample to be detected and the biological probe molecules on the biochip are contacted with each other, the two molecules are not specifically combined, so that the SPR response signal is kept unchanged as a signal curve B. The higher the concentration of the target molecule in the biological sample is, the larger the signal change of the SPR signal of the site where the target molecule and the biological sample are specifically combined is, namely the larger the signal difference R between the curve A and the curve B is, and the signal difference R is in a direct proportion relation with the concentration of the target molecule.
In a specific embodiment, as shown in fig. 5 and fig. 6, each sample channel unit 8 further includes a second sample inlet 82 and a second sample outlet 83, the second sample inlet 82 and the second sample outlet 83 are respectively communicated with the plurality of second microfluidic channels 81, after a biological sample is introduced into the sample channel unit 8 from the second sample inlet 82, the biological sample can be combined with the probe through a plurality of channels at the same time, and the biological sample repeatedly flows in the plurality of second microfluidic channels 81 between the second sample inlet 82 and the second sample outlet 83 by changing a pressure difference between the second sample inlet 82 and the second sample outlet 83, so that the combination efficiency of the probe molecules and the target molecules in the biological sample is improved through a plurality of times of reciprocating flows. In addition, the probe molecules can be fully contacted with the target molecules through the vibration of the whole multichannel biological reaction sensing device, and the combination efficiency of the probe molecules and the target molecules is improved.
In order to further improve the binding efficiency between the probe molecules and the target molecules in the biological sample, the inner wall of each second microfluidic channel 81 is provided with a saw-toothed channel structure, the saw-toothed channel structure is as shown in fig. 10, the saw-toothed channel structure comprises a plurality of saw-toothed channels, and the saw-toothed channels are arranged on the inner wall of the second microfluidic channel 81 at equal intervals. After the biological sample is respectively introduced into the second microfluidic channels 81 through the second sample inlets 82, the zigzag channel structure on the side walls of the second microfluidic channels 81 can make the biological sample generate turbulence locally when circulating in the second microfluidic channels 81, and change a stable microfluidic field at a local position, so that the combination efficiency of probe molecules and target molecules in the biological sample is improved, the signal response is enhanced, and the detection sensitivity is improved.
In summary, the present invention provides a multi-channel biological reaction sensor chip, and a method and an apparatus for manufacturing the same, comprising: the probe comprises a sensing chip body and a plurality of probe channels arranged on the sensing chip body, wherein different types of probe molecules are respectively fixed on the probe channels. According to the invention, through the combined action of the plurality of probe channels fixed with different types of probe molecules and the sample channel attached to the probe channels in an orthogonal relation, the SPRi detection can realize the specificity analysis and the content detection of various target molecules in a biological sample, and the sensing efficiency is improved. In addition, the structural design of the pipeline is avoided, the sample in the microfluidic channel is controlled through pressure difference, the detection sensitivity is improved, and the thought is provided for solving the problems that the SPRi is easy to pollute the biological sample, the operation is complex, the sample loss is large and the like and the clinical application of the future SPRi.
It is to be understood that the system of the present invention is not limited to the above examples, and that modifications and variations may be made by one of ordinary skill in the art in light of the above teachings, and all such modifications and variations are intended to fall within the scope of the appended claims.
Claims (10)
1. A multi-channel biological response sensor chip, comprising: the probe comprises a sensing chip body and a plurality of probe channels arranged on the sensing chip body, wherein different types of probe molecules are respectively fixed on the probe channels.
2. The multi-channel bioresponse sensing chip according to claim 1, wherein said sensing chip body comprises a glass substrate and a metal film disposed on said glass substrate.
3. The multi-channel bioreaction sensor chip of claim 1, wherein the probe channels are equally spaced apart on the sensor chip body and are parallel to each other.
4. A method for manufacturing a multi-channel biological reaction sensor chip according to any one of claims 1 to 3, comprising:
attaching a prefabricated mold to the surface of the sensing chip body; the mould comprises a plurality of concave parts, and when the mould is attached to the surface of the biochip body, a plurality of first microflow channels are formed between the concave parts and the biochip body;
respectively introducing a plurality of different types of probe molecule solutions into the plurality of first micro-flow channels, and enabling the plurality of different types of probe molecule solutions to be in contact with the surface of the biochip body through the plurality of first micro-flow channels to form a plurality of probe channels on the biochip body; the inner surfaces of the plurality of concave parts are in a sawtooth shape, and the flow mode of the probe molecule solution in the plurality of first microflow channels is a turbulent flow mode with non-uniform flow speed.
5. The method as claimed in claim 4, wherein the mold further comprises a plurality of first sample inlets and a plurality of first sample outlets, the plurality of first sample inlets and the plurality of first sample outlets correspond to the plurality of first micro-flow channels one-to-one, and each of the first micro-flow channels is communicated with the corresponding first sample inlet and first sample outlet of each of the first micro-flow channels.
6. The method as claimed in claim 5, wherein the step of introducing the plurality of different types of probe molecule solutions into the plurality of first micro flow channels comprises:
and adjusting the pressure difference between the first sample inlet and the first sample outlet corresponding to each first micro-flow channel, and controlling the probe molecule solutions of different types to flow back and forth in the first micro-flow channels by the pressure difference.
7. A multi-channel biological reaction sensor device, comprising the multi-channel biological reaction sensor chip of any one of claims 1 to 3.
8. The multi-channel bioresponse sensing device of claim 7, wherein said device further includes: and each sample channel unit is orthogonally attached to the probe channels.
9. The multi-channel biological reaction sensing device of claim 8, wherein each of the sample channel units comprises a second sample inlet, a plurality of second microfluidic channels, and a second sample outlet, and the second sample inlet and the second sample outlet are communicated with the plurality of second microfluidic channels; after the biological sample is introduced into the sample channel unit from the second sample inlet, the biological sample repeatedly flows in the second microfluidic channels by changing the pressure difference between the second sample inlet and the second sample outlet.
10. The multi-channel bioresponse sensing device of claim 9, wherein the side walls of each second microfluidic channel are provided with a saw-tooth channel structure.
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