TWI718053B - Linker of bioprobes - Google Patents

Abstract

A linker of bioprobes, suitable for immobilizing a bioprobe on a substrate of a sensor, includes having a carbon number of 6 or more of SH-(CH)n-NH2, SH-(CH)n-COOH, SH-(CH)n-SH, (OH)m-(CH)n-COOH or (OH)m-(CH)n-NH2, m, n is an integer greater than 1. Wherein, when the surface average roughness (Ra) of the substrate is greater than 250 nm, the coverage of the linker on the substrate is 40%-80%. The invention further discloses a linker of bioprobes, includes having a carbon number of less than 6 of SH-(CH)n-NH2, SH-(CH)n-COOH, SH-(CH)n-SH, (OH)m-(CH)n-COOH or (OH)m-(CH)n-NH2, m, n is an integer greater than 1. Wherein, when the surface average roughness (Ra) of the substrate is less than 250 nm, the coverage of the linker on the substrate is 65%-100%. The invention has the optimal carbon chain length of the linker and the coverage for the substrates of various roughnesses, and can greatly enhance the grasping ability of the electrochemical sensor for the detected object.

Description

Linker

The present invention relates to a linker compound, in particular to a linker of a biological probe.

In the production of immuno-type electrochemical biosensors, the number of bio-probes fixed on the surface of the chip is related to the sensitivity of the chip, and the bio-probes are fixed on the surface of the chip through the connector ( linker) do grafting. One end of the linker contains specific functional groups that bind to different chemical reagents or small molecules. Therefore, how to promote the effective connection and coverage of the linker on the surface of the chip is the first condition for making a biosensor. In the past, when the connector was connected and covered, it was mostly focused on the maximum connection coverage of the connector on the substrate surface. However, the actual situation is that even if the connector reaches the maximum connection coverage, there is no positive correlation between the amount of grasping of the target object by the wafer.

In order to solve the above problems, it is necessary to develop a linker with a specific carbon chain length to match the surface roughness of the biosensing chip and the coverage of the linker on the chip to obtain the maximum amount of grasping of the detected target.

The purpose of the present invention is to provide a linker of a biological probe, which has a specific carbon chain length and can obtain the maximum amount of grasping of the detected target.

The present invention provides a bio-probe linker to achieve the above-mentioned object, which is suitable for fixing the bio-probe on the chip substrate of the sensor, and contains SH-(CH)n with carbon number 6 or above. -NH2, SH-(CH)n-COOH, SH-(CH)n-SH, (OH)m-(CH)n-COOH or (OH)m-(CH)n-NH2, m and n are greater than An integer of 1. Wherein, when the average surface roughness (Ra) of the chip substrate is greater than 250 nm, the coverage of the connector on the chip substrate is 40%-80%.

The present invention also provides a linker for biological probes, which is suitable for fixing the biological probes on the chip substrate of the sensor, including SH-(CH)n-NH2, SH-(CH) with carbon number less than 6 ) n-COOH, SH-(CH)n-SH, (OH)m-(CH)n-COOH or (OH)m-(CH)n-NH2, m and n are integers greater than 1. Wherein, when the average surface roughness (Ra) of the chip substrate is less than 250 nm, the coverage of the connector on the chip substrate is 65%-100%.

Compared with conventional linkers of biological probes, the present invention has the following advantages. The present invention has the best carbon chain length linkers and coverage for substrates with different roughness, which can greatly improve the effect of electrochemical sensor chips on the target to be detected. The ability to grasp objects.

The present invention discloses a linker of a biological probe, which can greatly improve the grasping ability of an electrochemical sensor chip to a detected target.

Example 1:

The linker of the biological probe in the first embodiment of the present invention is suitable for fixing the biological probe on the chip substrate of the sensor, and includes SH-(CH) with carbon number 6 or above n-NH2, SH-(CH)n-COOH, SH-(CH)n-SH, (OH)m-(CH)n-COOH or (OH)m-(CH)n-NH2, m and n are An integer greater than 1. Wherein, when the average surface roughness (Ra) of the chip substrate is greater than 250 nm, the coverage rate of the connector on the chip substrate is 40%-80%, and the coverage rate is preferably 50%-70%.

The material of the chip substrate of the sensor of the present invention is silicon, glass, graphene, gold, platinum or polymer. Choose gold or platinum wafer substrates, suitable for pickling process for roughening treatment. The pickling solution can be surface treated with a ratio of 1 part of 98wt% sulfuric acid and 3 parts of 33wt% hydrogen peroxide to obtain specific roughness through different treatment times degree. The wafer substrates of other glass, ceramic or polymer materials can be roughened by mechanical polishing, chemical etching or chemical mechanical polishing.

The roughened wafer substrate is immersed in a linker solution dissolved in 99.8wt% absolute alcohol and placed at room temperature to modify the surface of the wafer substrate, and then use 1-ethyl-3-(3-dimethyl Aminopropyl) carbodiimide (EDC) or N-hydroxysuccinimide (NHS) performs functional group activation reaction on the surface of the modified wafer substrate. Subsequently, electrochemical methods can be used to measure changes in the redox characteristics of the wafer substrate surface, and the coverage of modified molecules can be calculated from the changes in current density.

The biological probe of the present invention is a biological probe of enzyme, protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) or ORSV antibody.

Example 2:

The linker of the biological probe in the second embodiment of the present invention is suitable for fixing the biological probe on the chip substrate of the sensor, and includes SH-(CH)n-NH2 with carbon number 6 or less SH-(CH)n-COOH, SH-(CH)n-SH, (OH)m-(CH)n-COOH or (OH)m-(CH)n-NH2, m and n are integers greater than 1 . Wherein, when the average surface roughness (Ra) of the chip substrate is less than 250 nm, the coverage rate of the connector on the chip substrate is 65%-100%, and the coverage rate is preferably 80%-100%.

The other technical content of the second embodiment of the present invention is the same as that of the first embodiment, including the material of the wafer substrate; the surface roughening treatment of the wafer substrate; the modification of the linker solution; and the functional group activation reaction on the surface of the wafer substrate. In addition, the types of bioprobes applicable are also the same.

The measured data of the linker of the biological probe of the present invention:

In the present invention, pure gold is used as the substrate, the surface roughness of the substrate is changed by pickling, and then the surface is modified with C ₃ H with a thiol group (-SH) at one end and a carboxyl group (-COOH) at one end. ₆ O ₂ , 3-Mercaptopropionic acid 3-MPA (3-Mercaptopropionic acid, MPA) and C ₁₁ H ₂₂ O ₂ S, 11-mercaptoundecanoic acid 11-MUA (11-Mercaptoundecanoic acid) are used as linkers The biological probe Odontoglossum ringspot virus antibody (ORSV antibody) is fixed on the surface to facilitate the capture of the subsequent target Odontoglossum ringspot virus (ORSV virus) as a practical example and measurement data.

Use 98wt% sulfuric acid and 33wt% hydrogen peroxide to process the Au substrate surface at a ratio of 1:3. The roughness changes due to the difference in processing time. The surface of the Au substrate is analyzed by atomic force microscopy (AFM) Roughness. As shown in Figure 1, the average roughness (Ra) of the untreated (before treatment) surface is 1.6 nm, and the relative height (Z range) is 11.7 nm. As shown in Figure 2, the average surface roughness (Ra) of the surface after 20 minutes of pickling treatment is 3.56 nm, and the relative height (Z range) is 57.7 nm.

Soak the roughened gold substrate in a solution of 3-mercaptopropionic acid (3-MPA) and 11-mercaptoundecanoic acid (11-MUA) dissolved in 99.8wt% absolute alcohol and place it at room temperature for the gold substrate The surface is modified, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N-hydroxysuccinimide (NHS) is used to modify the surface of the wafer substrate Perform functional group activation reaction, as shown in Figure 3.

After pickling treatment for 20 minutes, the surface roughness of the gold substrate was analyzed using an atomic force microscope after modification with MPA. As shown in Figure 4, the average surface roughness (Ra) is 1.78 nm and the relative height (Z range) is 26.1 nm. After pickling treatment for 20 minutes, the surface roughness of the gold substrate was analyzed using an atomic force microscope after modification with MUA. As shown in Figure 5, the average surface roughness (Ra) is 1.43 nm and the relative height (Z range) is 18.7 nm.

Subsequently, electrochemical methods are used to measure the change in the redox characteristics of the wafer substrate surface, and the coverage of the linker (modification molecule) is further calculated from the change in current density. As shown in Figure 6, both 3-MPA and 11-MUA will fill up the roughness of the gold substrate surface, and the active area reaction shows that the coverage of 11-MUA is about 94.7%, and the coverage of 3-MPA is about 20%.

Dissolve the bioprobe Odontoglossum ringspot virus antibody (ORSV antibody) in a buffer and drop it on the activated MPA and MUA after surface modification to form the bioprobe ORSV antibody and the linker end The peptide bond (-CO-NH-) is then immobilized on the surface of the Au Substrate, and then the ORSV virus solution of the same concentration is connected and covered with the antibody, as shown in Figure 7.

Finally, the scanning electron microscope was used to analyze the amount of ORSV virus grabbing on the surface of the gold substrate by SEM. The results are shown in Figures 8 to 9. The coverage rate is less than 80% of the short carbon chain 3-MPA linker with carbon number 6 or less. The crawling volume of ORSV virus (Figure 8) is less than the crawling volume of ORSV virus by linkers with a coverage rate greater than 80% (Figure 9), that is, linkers with carbon number 6 or less, the greater the coverage rate is for The more ORSV virus is crawled. When the coverage of the long carbon chain 11-MUA linker is greater than 80%, the crawling amount of the ORSV virus is >80%, as shown in Figure 10. The greater the coverage of the long carbon chain, the greater the crawling of the ORSV virus. the amount.

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Figures 1 to 2 show the AFM analysis of the surface roughness of the gold substrate. Figure 3 is a schematic diagram of the functional group activation reaction on the surface of the modified wafer substrate. Figures 4 to 5 show the surface roughness of the gold substrate after modification using an atomic force microscope. Figure 6 shows the coverage of the linker of the present invention. Figure 7 is a schematic diagram of the bioprobe forming a peptide bond (-CO-NH-) with the end of the linker and then immobilizing it on the Au Substrate surface. Figures 8 to 9 are scanning electron microscope analysis photos of the amount of ORSV captured by the 3-MPA linker. Figure 10 is a scanning electron microscope analysis of the amount of ORSV captured by the 11-MUA linker.

Claims (5)

A linker of a biological probe, suitable for fixing the biological probe on the chip substrate of the sensor, including: SH-(CH)n-NH2, SH-(CH)n-COOH, SH-(CH)n-SH, (OH)m-(CH)n-COOH or (OH)m- with carbon number 6 or less (CH) n-NH2, m and n are integers greater than 1; Wherein, when the average surface roughness (Ra) of the chip substrate is less than 250 nm, the coverage rate of the connector on the chip substrate is 65%-100%. The connector of the biological probe according to claim 1, wherein the material of the chip substrate of the sensor is silicon, glass, graphene, gold, platinum or polymer. The linker of the biological probe according to claim 1, wherein the wafer substrate is surface-treated with a ratio of 1 part of 98wt% sulfuric acid to 3 parts of 33wt% hydrogen peroxide, and a specific roughness is obtained through different treatment times. The linker of the biological probe according to claim 1, further comprising immersing the chip substrate in a linker solution dissolved in 99.8 wt% absolute alcohol and placing it at room temperature to modify the surface of the chip substrate before use 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N-hydroxysuccinimide (NHS) performs functional group activation reaction on the surface of the modified wafer substrate. The linker of the biological probe according to claim 1, wherein the biological probe is an enzyme, protein, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) or dentate plaque Biological probe for ORSV antibody.

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