CN111551607B - Biological array for detection and detection method thereof - Google Patents
Biological array for detection and detection method thereof Download PDFInfo
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
The invention provides a biological array for detection, which comprises a biological array base layer and a plurality of signal channel combinations arranged on the surface of the biological array base layer, wherein each signal channel combination detects a biological molecule; each signal path combination comprises at least a first sensing end and a second sensing end; a capture coating and a biomolecule capture layer added on the capture coating are coated on the surface of the biological array substrate between the first sensing end and the second sensing end, the biomolecule capture layer captures biomolecules, and nanoparticles with the biomolecule capture layer are arranged on the biomolecule capture surface; a detection signal is generated between the first sensing end and the second sensing end through the nano particles. The biological array provided by the application can realize multiple detection by using few detection samples once, is applicable to various types of biological molecules, improves the applicability of the biological array and the sensitivity of detection, reduces the complexity of operation and promotes the use of the biological array.
Description
Technical Field
The invention relates to the medical field, in particular to a biological array for detecting a biological molecular index and a detection method thereof.
Background
In the medical field, detection of biomolecular indicators in the human body is a fundamental and important test for diagnosing diseases or supervising the effectiveness of medical protocols for adjustment. With the progress of medical research, medical researchers have a deeper understanding of more and more diseases, and the complex interactive relationship between biomolecules in human bodies leads to the generation of diseases, so that the number and types of biomolecules to be detected for diagnosing related diseases are greatly improved. However, at present, a plurality of medical detection means aim at a certain biological molecule, or detection of a plurality of biological molecules is realized through high-flux large laboratory equipment, and the sensitivity and the accuracy are low, and the operation is complicated.
The traditional laboratory detection adopts a micro-pore plate, when in use, liquid samples of a human body are evenly distributed into each hole of the micro-pore plate, and each hole is filled with a corresponding reagent for detecting a specified biological molecule. The detection and detection method needs to collect more human body liquid samples. Recently, with the popularization and rapid development of microelectronic chip technology, such microwell plates are gradually replaced by biological arrays.
The biological array miniaturizes each detection and reagent of the traditional micropore lath and detects the detection on a small-sized structure; because of the small area, the amount of liquid sample used in the test can be small (about 2. Mu.L-500. Mu.L). In addition, the manufacturing technology of microelectronics and semiconductor industries is developed, reducing the manufacturing cost of the biological arrays. In view of the above, one of the major trends in the medical detection field is the popularization of biological arrays. The existing biological array detection is also in a preliminary development stage, such as the existing detection by using a DNA array, and the PCR technology is utilized to amplify the DNA, so that the trace target DNA can be specifically amplified by millions of times, thereby greatly improving the analysis and detection capability of DNA molecules, but the DNase has high cost and high error rate, and the detection is often misreported. The DNA amplification requires 20 to 40 cycles of cold and hot (65-95 degrees) temperature, adding additional temperature regulation functionality to the detection device, which limits the popularization and use of the bioarray.
Thus innovations in biological arrays are highly desirable to achieve more sensitive, accurate and easy-to-operate detection of biological arrays.
Disclosure of Invention
The invention aims to provide a biological array for detection and a detection method thereof, so as to realize detection which is more sensitive, more accurate and simpler to operate.
To solve the above problems, the present application provides a bio-array for multiplex detection that can provide more sensitive multiplex biomolecule detection than the prior art. The biological array comprises a biological array base layer and a plurality of signal channel combinations arranged on the surface of the biological array base layer, wherein each signal channel combination detects a biological molecule; each signal path combination comprises at least a first sensing end and a second sensing end; a capture coating and a biomolecule capture layer added to the capture coating are coated on the surface of the bio-array substrate between the first sensing end and the second sensing end, the biomolecule capture layer captures the biomolecules, and the captured biomolecules capture nanoparticles with the biomolecule capture layer on the surface; and a detection signal is generated between the first induction end and the second induction end through the captured nano particles.
Further, the first sensing end and the second sensing end are both set as electrodes, or the first sensing end and the second sensing end are both set as fiber tail ends, or the first sensing end and the second sensing end are respectively set as a light emitting end and a light receiving end.
Preferably, the surfaces of the first sensing end and the second sensing end are provided with anti-sticking coatings to prevent biomolecules from sticking to the sensing section.
Further, a distance P between the first sensing end and the second sensing end such that a quotient of P divided by a diameter D of the nanoparticle is not less than a percolation threshold T (i.e., P/D > =t); the percolation threshold T refers to the number of nanoparticles between the first sensing end and the second sensing end when the signal to noise ratio (signal to noise ratio) is 2.
Preferably, a blank channel combination is further arranged on the surface of the biological array base layer, the blank channel combination comprises at least a first sensing end and a second sensing end, and a noise signal is generated between the first sensing end and the second sensing end of the blank channel combination.
The application also provides a detection method of the biological array, which uses the biological array, wherein the biological array comprises a biological array base layer and a plurality of signal channel combinations arranged on the surface of the biological array base layer, and each signal channel combination detects a biological molecule; each signal path combination comprises at least a first sensing end and a second sensing end; a capture coating and a biomolecule capture layer added on the capture coating are coated on the surface of the biological array substrate between the first sensing end and the second sensing end, and the biomolecule capture layer captures the biomolecules; a nanoparticle having the biomolecule-capturing layer on the biomolecule-capturing surface; a detection signal is generated between the first induction end and the second induction end through the nano particles; the detection method comprises the following steps: s1, adding a sample to be detected to the surface of the biological array, and capturing biomolecules in the sample by a biomolecule capturing layer; s2, adding a detection reagent containing nano particles to the surface of the biological array, wherein a biomolecule capturing layer for capturing biomolecules is arranged on the surface of the nano particles; the biomolecules captured in S1 capture nanoparticles; s3, a detection signal is generated between the first induction end and the second induction end through the nano particles captured in the S2; and between S1, S2 and S3, respectively flushing the biological arrays by using buffer solution.
Further, in the step S3, the detection signal is generated by weakening the optical signal by the nanoparticles between the first sensing end and the second sensing end, and preferably, the nanoparticles are plastic nanoparticles.
Further, in the step S3, a detection signal is generated between the first sensing end and the second sensing end by enhancing an optical signal with nanoparticles, and preferably, the nanoparticles are glass nanoparticles or metal nanoparticles.
Further, in the step S3, the detection signal is generated by changing the electrical signal between the first sensing end and the second sensing end through nanoparticles, and preferably, the nanoparticles are metal nanoparticles.
Further, the step S2 includes: s201, adding a reagent containing nano particles to the surface of a biological array, wherein a biological molecule capturing layer for capturing biological molecules is arranged on the surface of the nano particles; the biomolecules captured in S1 capture nanoparticles;
and S202, adding a nanoparticle reinforcing reagent to the surface of the biological array, wherein ions in the nanoparticle reinforcing reagent are precipitated into molecules and accumulated on the surface of the nanoparticles, increasing the diameter of the nanoparticles, and flushing the biological array by using buffer solutions among S1, S201, S202 and S3.
Preferably, the nanoparticle is gold nanoparticle or silver nanoparticle, the nanoparticle reinforcing agent is silver reinforcing agent, and Ag in the nanoparticle reinforcing agent is subjected to silver precipitation chemical reaction + The ions precipitate into molecular Ag, accumulate on the surfaces of gold or silver nanoparticles, and increase the diameter of the nanoparticles.
The biological array for detection provided by the application is used for miniaturizing each detection and reagent of the microporous plate strip of the traditional laboratory and carrying out multiple detection on a structure with a small area; the required human body liquid sample is reduced, and the detection operation is more convenient. The application provides a biological array adds different biological molecule and catches the layer between the response end, detects different biological molecules, and the detection sample that disposable is few can realize multiple detection, is applicable to the disease detection of multiple category, has improved biological array's suitability, has reduced the complexity of operation, utilizes biological array's popularization and use. The detection method provided by the application uses the innovative biological array structure, breaks through in the aspects of sensitivity and accuracy, simultaneously makes the operation simpler and improves the applicability of the biological array detection method. In addition, the detection method provided by the application can also selectively amplify the detection signal so as to realize high signal-to-noise ratio according to the sensing principle of a signal channel during detection and improve the sensitivity and the accuracy of medical detection.
Drawings
In order to make the above and other objects of the present invention more comprehensible, preferred embodiments accompanied with figures are described in detail below:
FIG. 1 is a schematic illustration of a biological array for detection of example 1 of the present application;
FIG. 2 is a schematic cross-sectional view of the signal channels in the biological array of FIG. 1 in combination;
FIG. 3 is a schematic cross-sectional view of one of the signal path combinations 2 of FIG. 1 in use;
FIG. 4 is a schematic cross-sectional view of another signal path combination 2' of FIG. 1 in use
FIG. 5 is a schematic illustration of a biological array for detection of example 2 of the present application;
FIG. 6 is a schematic cross-sectional view of the hollow white signal channel combination 7 of FIG. 4;
FIG. 7 is a graph of concentration versus light intensity of biomolecules detected in example 3 of the present application.
FIG. 8 is a graph of concentration versus light intensity for biomolecules detected in example 4 of the present application.
Fig. 9 is a schematic cross-sectional view of a signal path combination 2 employing a selectively amplified signal scheme according to embodiment 6 of the present application.
FIG. 10 is a graph of concentration of biomolecules detected in example 6 of the present application versus current.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present specification, by describing the embodiments of the present invention with specific examples.
Example 1
As shown in fig. 1, the present embodiment provides a biological array for detection. Fig. 2 is a schematic cross-sectional view of a combination of signal channels in the biological array shown in fig. 1.
As shown in fig. 1 and 2, the bio-array for detection of the present embodiment includes a bio-array substrate 1 and a plurality of signal channel combinations 2 provided on the surface of the bio-array substrate 1, each signal channel combination 2 detecting one bio-molecule; each signal path combination 2 includes at least a first sensing end 31 and a second sensing end 32.
The surface of the bio-array substrate 1 between the first sensing tip 31 and the second sensing tip 32 is coated with a capture coating 41 and a bio-molecule capture layer 42 added to the capture coating 41. The biomolecule capturing layer 42 captures biomolecules to be detected, and the biomolecule capturing layer 42 captures nanoparticles 5 having the biomolecule capturing layer 42 on the biomolecule capturing surface; a detection signal is generated between the first sensing tip 31 and the second sensing tip 32 by the captured nanoparticle 5.
The materials of the capture coating 41 and the bio-array substrate 1 may be any one or a combination of the prior art, and the bio-molecule capture layer 42 may be set according to bio-molecules to be detected.
For example, the material of the surface of the bio-array substrate 1 may be silicon dioxide or oxidized silicon nitride. The capture coating 41 may be a silane-R for a silica surface, and the R group may be a carboxilic acid group, an amine group, etc., suitable for the next addition of the biomolecule capture layer 42. Detailed chemical reactions are generally known (see Bioconjugation Techniques, greg T Hermanson,2013, third edition, academic Press) for specific details of how to use R-groups to add a biomolecule capture layer 42.
The capture coating 41 may be added to the surface of the biological array substrate 1 by means of prior art techniques, which are well established, and the surface chemistry available to achieve the capture coatings of the present application is a wide variety, further exemplified in this embodiment but not limited thereto. For example, droplets of capture coating 41 material may be sprayed onto the biological array substrate 1 by a nano-spray mode printer or a conventional spoter printer. Different capture coatings 41 can be applied between the sensing ends of different signal channel combinations 2, thereby enabling the biological array to perform the function of detecting multiple biological molecules.
The biomolecule capture layer 42 may be DNA, RNA, aptamer, protein antibodies, protein antigens, glycoproteins, fats, and the like. The biomolecule capture layer 42 specifically recognizes and captures biomolecules in a human sample (e.g., urine, saliva, blood). For example, the biomolecule is a prostate specific antigen (Prostate Specific Antigen PSA) that is an indicator of cancer, such as prostate cancer, where the biomolecule capture layer 42 is an antibody directed against PSA (anti-PSA antibody).
The surface of the nanoparticle 5 in the detection reagent used in conjunction with the biological array is also added with a biomolecule capture layer 42. How to manufacture nanoparticles with a biomolecule capture layer 42 on the surface is generally known (see Bioconjugation Techniques, greg T Hermanson,2013, third edition, academic Press, anti-body-conjugated nanoparticles for therapeutic applications, cardoso MM et al Curr Med Chem 2012,19, pp 3103-27).
As shown in fig. 1, there are 12 signal channel combinations 2 in fig. 1 that are capable of detecting at least 12 different biomolecules. Each signal channel combination 2 comprises at least two sensing ends, wherein each sensing end is a sensor, and detection signals are generated between two adjacent sensing ends through captured nano particles 5.
The distance between the signal channel combinations 2 is L, which can also be regarded as the coverage of one signal channel combination 2. L is arranged such that the combination of signal channels for two different detection purposes does not affect each other, i.e. the two different biological monitors do not interfere with each other. For example, when the capture coating 41 is applied to the biological array substrate 1 by spraying droplets, L is greater than or equal to the minimum radius of the sprayed droplets.
The distance P between two adjacent sensing ends is such that the quotient of P divided by the diameter D of the captured nanoparticle 5 is not less than the percolation threshold T; the percolation threshold T refers to the number of nanoparticles between the two sensing ends when the signal-to-noise ratio is 2. The signal-to-noise ratio refers to the ratio of the signal value to the noise value. When the number of nanoparticles is less than the percolation threshold T, the signal-to-noise ratio is less than 2, where the signal is not clearly distinguishable from noise in the environment and is generally considered to be an unmeasurable signal.
As shown in fig. 2, in the detection, a "sandwich" method is used, in which the biomolecules 3 to be detected in the sample are captured by the biomolecule capture layer 42, and the captured biomolecules 3 capture the nanoparticles 5 having the biomolecule capture layer 42 on the surface. According to the bio-array of the present invention, the bio-molecules 3 may adsorb enough nano-particles 5 so that a detection signal is generated between the first sensing terminal 31 and the second sensing terminal 32 through the captured nano-particles 5.
As shown in fig. 3, the signal channel assembly 2 includes 2 sensing terminals, namely a first sensing terminal 31 and a second sensing terminal 32, for detecting a biomolecule 3, capturing a nanoparticle 5, and the first sensing terminal 31 and the second sensing terminal 32 generate a detection signal through the captured nanoparticle 5.
As shown in fig. 4, the signal channel assembly 2 'includes 3 sensing terminals, namely, a first sensing terminal 31, a second sensing terminal 32 and a third sensing terminal 33, respectively, for detecting two kinds of biomolecules, respectively, and capturing the nanoparticles 5 and 5', respectively, and only when both kinds of biomolecules are captured, a detection signal can be generated between the first sensing terminal 31, the second sensing terminal 32 and the third sensing terminal 33. The nanoparticles 5 and 5' may be the same or different.
The sensing end is a sensor and can be any one of the prior art. For example, the first sensing terminal 31 and the second sensing terminal 32 may each be provided as an electrode for detecting an electrical signal therebetween; or the first sensing end 31 and the second sensing end may be both set as optical fiber tail ends, and optical signals between the two ends are detected; or the first sensing terminal 31 and the second sensing terminal 32 may be respectively configured as a light emitting terminal and a light receiving terminal, and the second sensing terminal 32 may receive the light signal emitted from the first sensing terminal 31.
As a further optimization, as shown in fig. 2, the surfaces of the first sensing tip 31 and the second sensing tip 32 may be provided with a release coating 6. The release coating 6 is applied to the sensing end surface and is an insulating coating that prevents biomolecules from adsorbing or sticking to the sensing end. The surface coating technology is mature and the surface chemistry that can be used to achieve the release coatings of the present application is a wide variety and is further exemplified in this example, but not limited to these. For example, a metal electrode surface may be coated with a thio-polyethylene glycol (thio-PEG), wherein the thio chemistry is directed to the metal surface, while the PEG chemistry with a high molecular weight in excess of 1000 has the function of preventing biomolecules, cells from adsorbing on the sensing end surface. By the arrangement of the release coating 6, noise of the detection signal can be effectively reduced.
Example 2
As shown in fig. 5, the present embodiment provides a biological array for detection. The biological arrays in this embodiment are otherwise identical or similar to those in embodiment 1, and will not be described in detail herein. Unlike in embodiment 1, the bio-array in this embodiment further includes at least one group of blank channel combinations 7 provided on at least the surface of the bio-array substrate 1.
As shown in fig. 5 and 6, the blank channel combination 7 includes at least a first sensing end 31 and a second sensing end 32. The surface of the bio-array substrate 1 between the first sensing end 31 and the second sensing end 32 of the blank channel combination 7 is not coated with the capture coating 41, nor is the bio-molecule capture layer 42 provided. A noise signal is generated between the first sensing end 31 and the second sensing end 32 of the blank channel combination 7.
When the signal measuring device is used, the signal measured between the first sensing end 31 and the second sensing end 32 of the blank signal channel combination 7 is a signal read under the condition of no biological index, and the signal value or the signal intensity at the moment can be used as a noise value or a noise intensity and can be used as a reference standard when the signal channel combination 2 measures the signal.
Example 3
The present embodiment provides a bioarray detection method, using the bioarray for detection provided in embodiment 2, the detection method including:
s1, adding a sample to be detected to the surface of the biological array, and capturing biomolecules 3 in the sample by a biomolecule capturing layer 42;
s2, adding a detection reagent containing nano-particles 5 to the surface of the biological array, wherein a biomolecule capturing layer 42 for capturing biomolecules 3 is arranged on the surface of the nano-particles 5; the biomolecules captured in S1 capture the nanoparticles 5 in the detection reagent;
s3 a detection signal is generated between the first sensing end 31 and the second sensing end 32 by the nanoparticles 5 captured in S2.
Wherein the detection reagent containing the nano-particles 5 is a dispersion liquid of the nano-particles 5 in a buffer solution.
As a further optimization, between S1, S2 and S3, the biological arrays are rinsed with buffer solution, respectively. Namely, after the sample to be detected is added to the surface of the biological array for detection, the biological array is washed by using buffer solution; after the detection reagent is added to the biological surface for detection, the biological array is flushed with a buffer solution.
The buffer is one of the usual buffers in the prior art. Such as PBS (phosphate buffer saline), tris-HCl, etc.
Specifically, the bio-array used in the present embodiment, the sensing terminals are provided as a light emitting terminal and a light receiving terminal, respectively. The detection signal is generated by weakening the optical signal between the sensing ends through the nano particles 5. In this case, the nanoparticles 5 are light blocking nanoparticles, for example, the nanoparticles 5 are glass nanoparticles or plastic nanoparticles.
Upon detection, the biomolecule 3 is captured by the biomolecule capture layer 42, and the captured biomolecule 3 captures the light blocking nanoparticle 5 having the biomolecule capture layer 42 on the surface. The originally existing optical signal channels between the sensing ends are blocked by the captured light blocking nano particles 5, and detection signals are generated between the sensing ends through the weakened intensity of the optical signals.
In this example, PSA and VEGF were used as the biomolecules to be detected, respectively, and the control group was the signal value measured by the sensing end of the blank channel combination 7. The distance between the sensing ends is p=d×t.
The relationship between the concentration of the biomolecule detected in this example and the light intensity is shown in FIG. 7. As can be seen from FIG. 7, using the bioarray and detection method provided herein, biomolecules at femtomolar (10-15M) molecular concentration can be detected.
The biological array and the detection method provided by the application have higher sensitivity and accuracy, are convenient to operate, can realize multiple detection by using few detection samples once, and are suitable for popularization and use of medical detection.
Example 4
The present embodiment provides a bioarray detection method, using the bioarray for detection provided in embodiment 2, the detection method including:
s1, adding a sample to be detected to the surface of the biological array, and capturing biomolecules 3 in the sample by a biomolecule capturing layer 42;
s2, adding a detection reagent containing nano-particles 5 to the surface of the biological array, wherein a biomolecule capturing layer 42 for capturing biomolecules 3 is arranged on the surface of the nano-particles 5; the biomolecules captured in S1 capture nanoparticles 5;
s3 a detection signal is generated between the first sensing end 31 and the second sensing end 32 by the nanoparticles 5 captured in S2.
Wherein the detection reagent containing the nano-particles 5 is a dispersion liquid of the nano-particles 5 in a buffer solution.
As a further optimization, between S1, S2 and S3, the biological arrays are rinsed with buffer solution, respectively. Namely, after the sample to be detected is added to the surface of the biological array for detection, the biological array is washed by using buffer solution; after the detection reagent is added to the biological surface for detection, the biological array is flushed with a buffer solution.
The buffer is one of the usual buffers in the prior art. Such as PBS (phosphate buffer saline), tris-HCl, etc.
Specifically, the bio-arrays used in this embodiment have sensing ends each provided as an optical fiber tail end. The detection signal is generated by enhancing the optical signal between the sensing ends through the nano particles 5. In this case, the nanoparticles 5 are light-guiding nanoparticles 5, for example, the nanoparticles 5 are metal nanoparticles. At a distance P between the sensing ends, the propagation of light in air is weak, glass nanoparticles can be used to pass through completely, and metal nanoparticles can enhance the optical signal by surface enhanced raman scattering (Surface enhanced raman scattering SERS). The SERS effect of metal nanoparticles is mentioned in several documents, such as al methods,2014,6, 9116-9123.
At the time of detection, the biomolecule 3 in the sample is captured by the biomolecule capture layer 42, and the captured biomolecule 3 captures the light-guiding nanoparticle 5 having the biomolecule capture layer 42 on the surface in the detection reagent. By using the biological array provided by the application, the biomolecules 3 can adsorb enough light guiding nano particles 5, so that a complete light guiding signal channel is formed between the first sensing end 31 and the second sensing end 32 through the light guiding nano particles 5, and a detectable detection signal is generated.
In this example, PSA and VEGF were used as the biomolecules to be detected, respectively, and the control group was the signal value measured by the sensing end of the blank channel combination 7. The distance between the sensing ends is p=d×t.
The relationship between the concentration of the biomolecule detected in this example and the light intensity is shown in FIG. 7. As can be seen from FIG. 8, using the bioarray and detection method provided herein, biomolecules at femtomolar (10-15M) molecular concentration can be detected.
The biological array and the detection method provided by the application have higher sensitivity and accuracy, are convenient to operate, can realize multiple detection by using few detection samples once, and are suitable for popularization and use of medical detection.
Example 5
The embodiment provides a biological array detection method, which uses the biological array for detection provided by the application, and the detection method comprises the following steps:
s1, adding a sample to be detected to the surface of the biological array, and capturing biomolecules 3 in the sample by a biomolecule capturing layer 42;
s2, adding a detection reagent containing nano-particles 5 to the surface of the biological array, wherein a biomolecule capturing layer 42 for capturing biomolecules 3 is arranged on the surface of the nano-particles 5; the biomolecules captured in S1 capture nanoparticles 5;
s3 a detection signal is generated between the first sensing end 31 and the second sensing end 32 by the nanoparticles 5 captured in S2.
Wherein the detection reagent containing the nano-particles 5 is a dispersion liquid of the nano-particles 5 in a buffer solution.
As a further optimization, between S1, S2 and S3, the biological arrays are rinsed with buffer solution, respectively. Namely, after the sample to be detected is added to the surface of the biological array for detection, the biological array is washed by using buffer solution; after the detection reagent is added to the biological surface for detection, the biological array is flushed with a buffer solution.
The buffer is one of the usual buffers in the prior art. Such as PBS (phosphate buffer saline), tris-HCl, etc.
Specifically, the bio-arrays used in the present embodiment have sensing terminals each provided as an electrode. For example, the sensing tip may be a metal electrode, and more particularly, the sensing tip may be an inter-digital electrode (inter-digitated electrodes). The sensing terminals generate a detection signal, e.g. an increase current signal or a decrease voltage signal, by changing the electrical signal via the nanoparticles 5. In this case, the nanoparticles 5 are conductive nanoparticles 5, for example, the nanoparticles 5 are metal nanoparticles.
Upon detection, the biomolecule 3 in the sample is captured by the biomolecule capture layer 42, and the captured biomolecules 3 capture the conductive nanoparticles 5 having the biomolecule capture layer 42 on the surface in the detection reagent. With the biological array provided by the application, the biomolecules 3 can adsorb enough conductive nano particles 5, so that a complete conductive signal channel is formed between the first sensing end 31 and the second sensing end 32 through the conductive nano particles 5, and a detectable detection signal is generated.
Example 6
The present embodiment provides a method for detecting a biological array, using the biological array for detection provided in embodiment 2 of the present application, the method for detecting a biological array includes:
s1, adding a sample to be detected to the surface of the biological array, and capturing biomolecules 3 in the sample by a biomolecule capturing layer 42;
s201, adding a detection reagent containing nano-particles 5 to the surface of a biological array, wherein a biomolecule capturing layer 42 for capturing biomolecules 3 is arranged on the surface of the nano-particles 5; the biomolecules captured in S1 capture nanoparticles 5;
s202, adding a nano particle reinforced reagent to the surface of the biological array, wherein ions in the nano particle reinforced reagent are precipitated into molecules and accumulated on the surface of the nano particle 5, so that the diameter of the nano particle 5 is increased;
s3 a detection signal is generated between the first sensing end 31 and the second sensing end 32 by the nanoparticles 5 captured in S2. The detection signal is generated by enhancing the electrical signal (current) between the first sensing terminal 31 and the second sensing terminal 32 through the nanoparticles 5.
Wherein the nano particles 5 are gold nano particles or silver nano particles, the nano particle reinforcing reagent is silver reinforcing reagent, and Ag in the nano reinforcing reagent is subjected to silver precipitation chemical reaction + The ions are precipitated into molecular Ag, and are accumulated on the surfaces of gold or silver nano-particles 5, so that the diameters of the nano-particles 5 are increased;
wherein the detection reagent containing the nano-particles 5 is a dispersion liquid of the nano-particles 5 in a buffer solution.
As a further optimization, between S1, S201, S202 and S3, the biological arrays are rinsed with buffer solution, respectively. Namely, after the sample to be detected is added to the surface of the biological array for detection, the biological array is washed by using buffer solution; after the detection reagent is added to the biological surface for detection, the biological array is flushed with a buffer solution.
The buffer is one of the usual buffers in the prior art. Such as PBS (phosphate buffer saline), tris-HCl, etc.
Specifically, the bio-arrays used in the present embodiment have sensing terminals each provided as an electrode. For example, the sensing tip may be a metal electrode, and more particularly, the sensing tip may be an inter-digital electrode (inter-digitated electrodes). The sensing terminals generate a detection signal, e.g. an increase current signal or a decrease voltage signal, by changing the electrical signal via the nanoparticles 5. The nanoparticle 5 is gold nanoparticle or silver nanoparticle.
This embodiment uses a silver enhancing agent to increase the diameter of the nanoparticle captured between the sensing ends, a way to selectively amplify the signal. A schematic cross-sectional view of a signal path combination 2 according to this embodiment is shown in fig. 9. When the nanoparticle 5 captured between the first sensing end 31 and the second sensing end 32 is smaller than the percolation threshold T, a detection signal may be generated by increasing the diameter of the nanoparticle 5.
As shown in fig. 9, when the distance P between the sensing ends of the bio-array is fixed and the content of the bio-molecules in the sample is very low, the metal nano-particles adsorbed on the surface of the bio-array substrate 1 are very small and insufficient to reach the permeation threshold T, at this time, no detection signal can be generated between the sensing ends, the bio-molecules with very low content cannot be detected, and the detection result will show that the sample does not contain the bio-molecules to be detected. At this time, the diameter of the extremely small metal nanoparticles captured between the sensing terminals can be increased using the "selective amplification signal method". The metal nano particles are silver nano particles or gold nano particles, and when the silver reinforcing reagent is added to the surface of the biological array, the gold nano particles or silver nano particles captured between the sensing ends catalyze Ag in the silver reinforcing reagent + ->Chemical reaction of Ag to react Ag in the reagent + The metallic silver is precipitated and deposited on the surface of the nano-particles, so that the diameter of the nano-particles is increased, and the diameter is increased from the original diameter D to a new diameter D'. At this time, t=p/D ', the diameter of the nanoparticle is changed from D to D', and the diameter is increased, so that the percolation threshold T is reduced, so that the metal nanoparticle adsorbed between the sensing ends may exceed the percolation threshold, and a detectable signal may be generated between the sensing ends. The method provided by the embodiment can be used for detecting a very small amount of organismsMolecules that increase the sensitivity of the biological array. Such a method of silver reinforcing nanoparticles is mentioned in the relevant literature, for example, talanta,148 (2016), 272-278.
By silver precipitation chemical reaction, the actual volume of the nano particles is amplified, the detection signal can be enhanced, and a small amount of biomolecules can be detected in a super-sensitive way. Silver precipitation chemical reaction Ag + ->The Ag technology can lift the nano particles with the diameter of 40 nanometers to 1.2 micrometers in 10 minutes, and can be amplified by 30 times, and can be operated at normal temperature. The method provided by the application is quick, simple, reliable and low in cost, and improves the sensitivity of biomolecule detection.
In this example, PSA and VEGF were used as the biomolecules to be detected, respectively, and the control group was the signal value measured by the sensing end of the blank channel combination 7. The distance between the sensing ends is p=d×t, and the silver enhancing reagent is a commercially available kit (e.g., ab170733, L-24919, etc. from Abcam corporation).
In the experiment, when step S201 is completed, no signal is detected between the sensing terminals, and after step S202 is completed, the silver-enhanced reagent is applied to the surface of the biological array for 15min, detection is performed, and the relationship between the detected current signal and the concentration of the biological molecule between the sensing terminals is shown in fig. 10. As can be seen from FIG. 10, using the bioarray and detection method provided herein, biomolecules at femtomolar (10-15M) molecular concentration can be detected.
According to the biological array and the detection method, multiple detection can be realized by using few detection samples once, the biomolecules which are originally extremely small in content and cannot be detected are detected by using a selective signal amplification mode, the sensitivity and the accuracy of biological array detection are improved, the operation is convenient, and the biological array detection method is suitable for popularization and use of medical detection.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (4)
1. A biological array for detection, characterized in that the biological array comprises a biological array substrate (1) and a plurality of signal channel combinations (2) arranged on the surface of the biological array substrate (1), wherein each signal channel combination (2) can detect at least one biological molecule (3);
each signal path combination (2) comprises at least a first sensing end (31) and a second sensing end (32);
a capture coating (41) and a biomolecule capture layer (42) added to the capture coating (41) are coated on the surface of the biological array substrate (1) between the first sensing end (31) and the second sensing end (32), the biomolecule capture layer (42) captures the biomolecules (3), and the captured biomolecules (3) capture nanoparticles with the biomolecule capture layer (42) on the surface;
the at least one signal channel combination (2) further comprises a third sensing end (33), a capture coating and a biomolecule capture layer added to the capture coating are coated on the surface of the bio-array substrate between the second sensing end (32) and the third sensing end (33), the biomolecule capture layer captures another biomolecule, and the captured nanoparticle with the biomolecule capture layer on the surface of the other biomolecule capture;
-the first sensing end (31) and the second sensing end (32), and-the distance (P) between the second sensing end (32) and the third sensing end (33) is larger than the diameter of the biomolecule;
the first sensing end (31), the second sensing end (32) and the third sensing end (33) are used for respectively detecting two biomolecules and respectively capturing nano particles, and when both the biomolecules are captured, detection signals are generated between the first sensing end (31), the second sensing end (32) and the third sensing end (33).
2. The biological array for detection according to claim 1, wherein the first sensing end (31) and the second sensing end (32) are each provided as an electrode, or the first sensing end (31) and the second sensing end (32) are each provided as an optical fiber tail end, or the first sensing end (31) and the second sensing end (32) are respectively provided as a light emitting end and a light receiving end; the surfaces of the first sensing end (31) and the second sensing end (32) can be provided with a release coating (6).
3. The biological array for detection according to claim 1, characterized in that the distance P between the first sensing end (31) and the second sensing end (32) is such that the quotient of P divided by the diameter D of the nanoparticle (5) is not less than the penetration threshold T; the percolation threshold T refers to the number of nanoparticles between the first sensing end (31) and the second sensing end (32) when the signal to noise ratio is 2.
4. The biological array for detection according to claim 1, wherein at least one group of blank channel combinations (7) is further provided on the surface of the biological array substrate (1), the blank channel combinations comprising at least a first sensing end (31) and a second sensing end (32), and noise signals are generated between the first sensing end (31) and the second sensing end (32) of the blank channel combinations (7).
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