CN111088331B - A Single-Molecule Sequencing Method Based on Piezoelectric Acoustic Sensor - Google Patents

Abstract

The invention discloses a single-molecule sequencing method based on a piezoelectric acoustic wave sensor, comprising the following steps: S1. modifying DNA polymerase on the surface of the piezoelectric acoustic wave sensor; Amplification principle Modifies the magnetic beads at the active end of the nucleotide phosphate chain; S4. Injection of the modified nucleotide; S5. Applying a magnetic field on the other side of the micropore of the acoustic wave sensor; S6. Elution on the surface of the sensor: S7. Test The frequency signal f ₁ of the acoustic wave sensor; S8. Using DNA polymerase to remove the active end modified magnetic beads of the nucleotide phosphate chain: S9. Testing the frequency signal f ₂ of the acoustic wave sensor; calculating the difference between f ₁ and f ₂ , Determine the base type of the single strand of the DNA template; S10. Clean the flow channel; repeat the above steps S3-S10 to perform continuous sequencing on the single strand of the DNA template in the microwell; it improves the detection sensitivity and reduces the sequencing cost.

Description

A Single Molecule Sequencing Method Based on Piezoelectric Acoustic Sensor

technical field

The invention relates to the field of DNA sequencing, in particular to a single-molecule sequencing method.

Background technique

Gene sequencing (Gene sequencing, or translation gene sequencing) refers to the analysis of the base sequence of a specific gene fragment, that is, the arrangement of adenine (A), thymine (T), cytosine (C) and guanine (G) Way. The advent of rapid gene sequencing methods has greatly facilitated research and discovery in biology and medicine.

Existing gene sequencing methods include optical method (array hole) and electrochemical method (single hole), and the array hole optical method requires nanopores to suppress signal noise, and the complexity and size of the system lead to high sequencing costs. It is necessary to propose a sequencing method Inexpensive, high-sensitivity sequencing method.

Contents of the invention

In view of the deficiencies in the prior art, the main purpose of the present invention is to provide a single-molecule sequencing method based on a piezoelectric acoustic wave sensor, which has high detection sensitivity and reduces the cost of gene sequencing.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

A single-molecule sequencing method based on a piezoelectric acoustic wave sensor, comprising the following steps:

S1: Modification of DNA polymerase on the surface of piezoelectric acoustic wave sensor;

Immobilizing the DNA polymerase on the surface of the piezoelectric acoustic wave sensor by modifying the DNA polymerase on the surface of the piezoelectric acoustic wave sensor;

S2: DNA template single-stranded small fragment-driven injection:

Construct a single-stranded DNA library, and drive the obtained DNA template single-stranded small fragments into the micropores of the piezoelectric acoustic wave sensor to ensure that there is at most one DNA template single-stranded small fragment in a microwell, and the DNA template single-stranded fragments entering the microwells The surface of the acoustic wave sensor is combined with DNA polymerase;

S3: Based on the principle of mass amplification, the magnetic beads are modified at the active end of the nucleotide phosphate chain;

Modify the magnetic beads at the active ends of the four nucleotide phosphate chains to amplify the quality of the four nucleotides;

S4: Modified nucleotide injection:

Add four kinds of nucleotides that modify the magnetic beads into the micropores of the piezoelectric acoustic wave sensor;

S5: applying a magnetic field on the other side of the micropore of the acoustic wave sensor to drive the magnetic bead-modified nucleotide to move to the surface of the piezoelectric acoustic wave sensor;

A magnetic field is applied near the input electrode and output electrode of the piezoelectric acoustic wave sensor. Under the action of the magnetic field, the four nucleotides modified with magnetic beads move to the single strand of the DNA template, and the nucleotides with suitable bases are adsorbed on the piezoelectric acoustic wave sensor. Under the catalysis of DNA polymerase, it is complementary to the corresponding base of the DNA template single strand;

S6: Elution from the sensor surface:

When the magnetic field is removed, the nucleotides modified by the magnetic beads that have not undergone specific reactions are no longer adsorbed on the surface of the sensor, and the surface is washed with a buffer to make the nucleotides that have non-specific reactions leave the sensitive area of the sensor;

S7: testing the frequency signal of the acoustic wave sensor, marked as f ₁ ;

f ₁ is the frequency value after the DNA template single strand and the nucleotide complementary pairing reaction of the modified magnetic beads;

S8: Magnetic beads modified at the active end of the nucleotide phosphate chain by DNA polymerase:

During the synthesis process, the DNA polymerase excises the modified magnetic beads and releases the active end of the phosphate chain;

S9: testing the frequency signal of the acoustic wave sensor, marked as f ₂ ;

_f2 is the frequency value after the surface quality of the piezoelectric acoustic wave sensor changes after the magnetic beads are excised by DNA polymerase;

Calculating the difference between _f1 and _f2 , and determining the base type of the nucleotide reacting with the DNA template single strand through the difference, thereby determining the base type of the corresponding DNA template single strand;

S10: cleaning the flow channel;

Clean the flow channel with cleaning solution to wash away the magnetic beads removed by DNA polymerase;

S11: Repeat the above steps S3-S10 to perform continuous sequencing on the DNA template single strands in the microwells.

Further, the method of applying the magnetic field in the step S5 is to set a magnet near the input electrode and the output electrode of the sensor through the driving device, so that there is a small distance between the magnet and the piezoelectric film.

Preferably, the magnet is a permanent magnet.

Further, in step S1, the DNA polymerase is modified on the surface of the piezoelectric acoustic wave sensor by means of a specific reaction between the polymerase group and the group modified on the sensor surface and then binding.

Further, in step S1, biotin and avidin are used to respectively modify the surface of the sensor and the surface of the polymerase to promote the combination of the two to modify the DNA polymerase on the surface of the piezoelectric acoustic wave sensor.

Further, in step S4, the four nucleotides of the modified magnetic beads are introduced into the microwells by means of microfluidics, specifically, the modified nucleotides are introduced into the microwells by a peristaltic pump or a syringe pump.

Preferably, the washing solution used in step S10 is PBS buffer.

Further, the piezoelectric acoustic wave sensor includes a piezoelectric thin film layer, a silicon layer, a silicon dioxide layer and an SOI silicon layer arranged in sequence, and several micropores are arranged on the silicon dioxide layer and the SOI silicon layer, and the The other side of the electric film layer is provided with an input electrode, an output electrode and a frequency signal collecting device.

Preferably, the material of the input electrode and the output electrode can be gold or aluminum; the material of the piezoelectric film layer can be aluminum nitride, piezoelectric ceramics or zinc oxide.

Preferably, the micropores are square or circular. When the micropores are square, their side length is 100 μm to 1000 μm. When the micropores are circular, their diameter is 100 μm-1000 μm; cloth.

One of the above technical solutions has the following advantages or beneficial effects: the piezoelectric acoustic wave sensor is used for single-molecule sequencing, and the detection sensitivity is improved by amplifying the quality of nucleotides, and the use of acoustic waves does not require the use of nanometer sensors to suppress signal noise. Holes avoid signal distortion caused by interference from optical factors, and do not require complex and bulky optical systems, resulting in low sequencing costs.

Description of drawings

FIG. 1 is a schematic diagram of a single-molecule sequencing based on a piezoelectric acoustic wave sensor proposed according to an embodiment of the present invention;

Fig. 2 is a structural schematic diagram of a piezoelectric acoustic wave sensor proposed according to an embodiment of the present invention;

Fig. 3 is a flow chart of performing single-molecule sequencing by a gene piezoelectric acoustic wave sensor proposed according to an embodiment of the present invention;

FIG. 4 is an enlarged view of the single-molecule sequencing structure diagram of the piezoelectric acoustic wave sensor in part A of FIG. 1 proposed according to an embodiment of the present invention.

In the figure: 1. Input electrode, 2. Output electrode, 3. Piezoelectric film layer, 4. Silicon layer, 5. Silicon dioxide layer, 6. SOI silicon layer, 7. Frequency signal acquisition device, 8. DNA Polymerase, 9, DNA template single strand, 10, primer, 11, nucleotide, 12, magnetic bead, 13, microwell.

Detailed ways

Below in conjunction with accompanying drawing, the present invention will be described in further detail, so that those skilled in the art can implement according to referring to description.

It should be understood that terms such as "having", "comprising" and "including" as used herein do not entail the presence or addition of one or more other elements or combinations thereof.

In the drawings, the shapes and dimensions may be exaggerated for clarity, and the same reference numerals will be used throughout to designate the same or like parts.

According to an embodiment of the present invention, with reference to FIGS. 1-2 , the single-molecule sequencing method based on the piezoelectric acoustic wave sensor of the present invention provides a piezoelectric acoustic wave sensor. The piezoelectric acoustic wave sensor includes a piezoelectric thin film layer 3 and also includes a The silicon wafer layer 4, the silicon dioxide layer 5 and the SOI silicon wafer layer 6 arranged in sequence on one side of the piezoelectric film layer 3, the silicon dioxide layer 5 and the SOI silicon wafer layer 5 on the other side of the silicon wafer layer 4 by a gas etching method A plurality of micropores 13 isolated from each other and arranged in an array are formed on the sheet 6. The micropores are square or circular. When the micropores are square, their side length is 100 μm to 1000 μm. -1000μm, the reaction solution is placed in the micropore; the input electrode 1, the output electrode 2 and the frequency signal acquisition device 7 are arranged on the other side of the piezoelectric film layer 3, and the material of the input electrode 1 and the output electrode 2 is gold (Au) or aluminum (Al), the piezoelectric film layer 3 may be aluminum nitride (AlN), piezoelectric ceramics (PZT) or zinc oxide (ZnO).

When the quality of the substance adsorbed on the surface of the piezoelectric acoustic wave sensor changes more obviously, the greater the change of its resonant frequency. That is, the frequency change of the piezoelectric acoustic wave sensor is affected by the change of the adsorption mass. Since the mass of a single nucleotide 11 is small, the mass of its adsorption near the surface of the sensor does not change much, and the measured frequency does not change significantly. Therefore, this embodiment modifies the magnetic bead 12 on the nucleotide 11 based on the principle of mass amplification, that is, the amplification core The mass of nucleotide 11. Under the action of an external magnetic field, the nucleotides 11 of the modified magnetic beads 12 with suitable bases are adsorbed to the surface of the sensor and react with the single strand 9 of the DNA template. At this time, the equivalent adsorption mass on the surface of the acoustic wave sensor is relatively large, and a frequency is measured Information, when the reaction is over, DNA polymerase 8 is used to excise the magnetic beads 12 modified by the nucleotide 11. At this time, the surface quality of the acoustic wave sensor is relatively small, and then a frequency information is measured. Since the surface quality of the acoustic wave sensor changes significantly, the measured The obtained frequency changes will also be more obvious, and the base type of the nucleotide 11 participating in the complementary pairing reaction can be determined by the frequency difference between the two, so as to deduce the base type of the corresponding DNA template single strand 9.

With reference to Figures 3-4, the present invention provides a single-molecule sequencing method based on a piezoelectric acoustic wave sensor, comprising the following steps:

S1: modifying DNA polymerase 8 on the surface of piezoelectric acoustic wave sensor;

The DNA polymerase 8 is immobilized on the surface of the piezoelectric acoustic wave sensor by modifying the DNA polymerase 8 on the surface of the piezoelectric acoustic wave sensor; The reaction is further combined; the other uses biotin and avidin to respectively modify the surface of the sensor and the surface of the polymerase to promote the combination of the two;

S2: DNA template single-stranded small fragment-driven injection:

Construct a single-stranded DNA library, drive the obtained DNA template single-stranded 9 small fragments into the microwell, ensure that there is at most one DNA template single-stranded 9 small fragment in a microwell, and the DNA template single-stranded 9 small fragments entering the microwell are in the piezoelectric acoustic wave sensor Surface binding to DNA polymerase 8;

S3: Based on the principle of mass amplification, the magnetic beads are modified at the active end of the nucleotide phosphate chain;

Modifying the magnetic beads 12 at the active ends of the four nucleotide phosphate chains is equivalent to amplifying the mass of the four nucleotides 11;

S4: Injection of modified nucleotide 11:

Adding four kinds of nucleotides 11 that modify the magnetic beads 12 into the micropores of the piezoelectric acoustic wave sensor;

The modified four nucleotides 11 can be introduced into the micropores by drop coating; they can also be introduced into the micropores by microfluidics, specifically by introducing the modified nucleotides into the micropores through a peristaltic pump or a syringe pump;

S5: applying a magnetic field on the other side of the micropore of the acoustic wave sensor to drive the nucleotide 11 modified by the magnetic bead 12 to move to the surface of the piezoelectric acoustic wave sensor;

A magnet is set near the input electrode 1 and the output electrode 2 of the sensor through the driving device to apply a magnetic field, thereby adsorbing the nucleotide 11. Specifically, the permanent magnet is selected. Since there is a reaction liquid in the micropore, there must be a small gap between the permanent magnet and the film. interval.

Under the action of a magnet set near the sensor electrodes, the four nucleotides 11 of the modified magnetic beads 11 move to the single strand 9 of the DNA template and are adsorbed on the surface of the piezoelectric acoustic wave sensor, and the nucleotides 11 with suitable bases are polymerized in the DNA Complementary pairing with the corresponding base of DNA template single strand 9 under the action of enzyme 8;

S6: Elution from the sensor surface:

Remove the magnetic field, and specifically remove the magnet through the driving device. At this time, the fragments that have not undergone specific magnetic bead modification are no longer adsorbed on the surface of the sensor, and the peristaltic pump or syringe pump is used to drive the buffer to wash the surface, so that the nucleosides with non-specific reactions Acid leaves the sensitive area of the sensor;

S7: testing the frequency signal of the acoustic wave sensor, marked as f ₁ ;

f ₁ is the frequency after the DNA template single strand and the nucleotide complementary pairing reaction of the modified magnetic beads. At this time, the equivalent adsorption mass on the sensor surface is relatively large;

S8: DNA polymerase 8 is used to excise the active end of the nucleotide phosphate chain modified magnetic beads 12 (ie, the mass amplification part):

Referring to the dotted line in Figure 4, during the synthesis process, DNA polymerase 8 can excise the modified magnetic beads 12 and release the active end of the phosphate chain;

S9: testing the frequency signal of the acoustic wave sensor, marked as f ₂ ;

When the magnetic bead 12 is excised by the DNA polymerase 8, the mass on the surface of the piezoelectric acoustic wave sensor changes, so the resonance frequency changes, and the changed frequency value is defined as f ₂ .

Calculating the difference between _f1 and _f2 , and determining the base type of nucleotide 11 reacting with the DNA template single strand 9 through the difference, thereby determining the base type of the corresponding DNA template single strand 9;

S10: cleaning the flow channel;

Wash the flow channel with conventional PBS buffer, etc., and wash away the magnetic beads excised by DNA polymerase 8;

S11: repeat the above steps S3-S10, sequentially detect the bases on the single strand of the DNA template,

End the sequencing process until the chain length reaches 200-5000 bp.

As mentioned above, the present invention uses piezoelectric acoustic wave sensors for single-molecule sequencing, and by amplifying the quality of nucleotides, the detection sensitivity is improved. The use of acoustic waves does not require nanopores, avoids signal distortion caused by optical factors, and does not require Complex and bulky optical system, low cost of sequencing.

The number of devices and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations to the piezoelectric acoustic wave sensor-based single-molecule sequencing method of the present invention will be apparent to those skilled in the art.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Further modifications can be effected, so the invention is not limited to the specific details and examples shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims (10)

1. A unimolecular sequencing method based on a piezoelectric acoustic wave sensor is characterized in that: the method comprises the following steps:

s1: modifying DNA polymerase on the surface of the piezoelectric acoustic wave sensor;

fixing the DNA polymerase on the surface of the piezoelectric acoustic wave sensor by modifying the DNA polymerase on the surface of the piezoelectric acoustic wave sensor;

s2: DNA template single-chain small fragment driving sample injection:

constructing a single-chain DNA library, driving the obtained DNA template single-chain small fragments to enter the micropores of the piezoelectric acoustic wave sensor, ensuring that at most one DNA template single-chain small fragment exists in one micropore, and combining the DNA template single chains entering the micropores with DNA polymerase on the surface of the piezoelectric acoustic wave sensor;

s3: modifying magnetic beads at the active ends of the nucleotide phosphate chains based on the mass amplification principle;

modifying magnetic beads at the active ends of the four nucleotide phosphate chains to amplify the mass of the four nucleotides;

s4: injecting modified nucleotide:

adding four nucleotides for modifying magnetic beads into micropores of the piezoelectric acoustic wave sensor;

s5: applying a magnetic field on the other side of the micropore of the acoustic wave sensor to drive the magnetic bead modified nucleotide to move towards the surface of the piezoelectric acoustic wave sensor;

applying a magnetic field near an input electrode and an output electrode of the piezoelectric acoustic sensor, moving four nucleotides of the modified magnetic beads to the DNA template single strand under the action of the magnetic field, adsorbing the nucleotides with proper bases on the surface of the piezoelectric acoustic sensor, and complementarily pairing the nucleotides with the corresponding bases of the DNA template single strand under the catalytic action of DNA polymerase;

s6: and (3) eluting the sensor surface:

removing the magnetic field, wherein the nucleotide modified by the magnetic beads without specific reaction is not adsorbed on the surface of the sensor any more, and washing the surface by adopting a buffer solution to separate the nucleotide with non-specific reaction from a sensitive area of the sensor;

s7: measuring the frequency signal of the acoustic wave sensor, denoted f ₁ ；

f ₁ Is the frequency value after the complementary pairing reaction of the DNA template single strand and the nucleotide of the modified magnetic bead;

s8: magnetic beads modified with active ends that cleave the nucleotide phosphate chains with DNA polymerase:

in the synthesis process, the modified magnetic beads are cut off by DNA polymerase, and the active end of the phosphate chain is released;

s9: measuring the frequency signal of the acoustic wave sensor, denoted f ₂ ；

f ₂ The frequency value is the frequency value after the surface quality of the piezoelectric acoustic wave sensor is changed after the magnetic beads are cut off by DNA polymerase;

calculating f ₁ And f ₂ And determining the base type of the nucleotide reacting with the single strand of the DNA template by the difference, thereby determining the base type of the corresponding single strand of the DNA template;

s10: cleaning the flow channel;

cleaning the flow channel by using a cleaning solution, and washing away the magnetic beads cut off by the DNA polymerase;

s11: repeating the steps S3-S10, and continuously sequencing the DNA template single strand in the micropore.

2. The method of claim 1, wherein: in the step S5, the magnetic field is applied by providing a magnet near the sensor input electrode and the sensor output electrode by a driving device so that the magnet and the piezoelectric thin film have a minute gap therebetween.

3. The method of claim 2, wherein: the magnet is a permanent magnet.

4. The method of claim 1, wherein: in the step S1, DNA polymerase is modified on the surface of the piezoelectric acoustic wave sensor in a mode of carrying out specific reaction and then combining a polymerase group and a group modified on the surface of the sensor.

5. The method of claim 1, wherein: in step S1, DNA polymerase is modified on the surface of the piezoelectric acoustic wave sensor in a manner that biotin and avidin are respectively modified on the surface of the sensor and the surface of polymerase to promote the combination of the sensor and the polymerase.

6. The method of claim 1, wherein: in the step S4, the four nucleotides of the modified magnetic beads are introduced into the micropores in a microfluidic mode, and the modified nucleotides are specifically introduced into the micropores through a peristaltic pump or an injection pump.

7. The method of claim 1, wherein: the washing solution used in step S10 is PBS buffer.

8. The method of claim 1, wherein: the piezoelectric acoustic wave sensor comprises a piezoelectric film layer, a silicon wafer layer, a silicon dioxide layer and an SOI silicon wafer layer which are sequentially arranged, a plurality of micropores are formed in the silicon dioxide layer and the SOI silicon wafer layer, and an input electrode, an output electrode and a frequency signal acquisition device are arranged on the other side of the piezoelectric film layer.

9. The method of claim 8, wherein: the input electrode and the output electrode are made of gold or aluminum; the piezoelectric film layer is made of aluminum nitride, piezoelectric ceramics or zinc oxide.

10. The method of claim 1, wherein: the micropores are square or round, the side length of the micropores is 100 mu m to 1000 mu m when the micropores are square, and the diameter of the micropores is 100 mu m to 1000 mu m when the micropores are round; the micropores are isolated from each other and arranged in an array.

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