CN109870433B

CN109870433B - Buoyancy microsphere probe for quantitative screening of ganglioside and preparation method thereof

Info

Publication number: CN109870433B
Application number: CN201711272434.7A
Authority: CN
Inventors: 鞠熀先; 陈云龙; 刘惠普
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2017-12-01
Filing date: 2017-12-01
Publication date: 2021-06-15
Anticipated expiration: 2037-12-01
Also published as: CN109870433A

Abstract

The invention relates to a pair of buoyancy microsphere probes for quantitatively screening cell surface ganglioside and a preparation method thereof. The buoyant microsphere probe comprises an extraction probe and a collection probe. The extraction probe takes a silica buoyancy microsphere modified by maleimide as a substrate, and is covalently bonded with a DNA molecule with an endonuclease cutting site and a boric acid terminal. Through covalent recognition of boric acid and sialic acid on the cell surface, the extraction probe can extract biomolecules containing sialic acid on the cell surface through buoyancy. The extracted biomolecules can be released into the solution under the action of endonuclease, and the ganglioside with the nucleic acid fragments combined therein is collected by a collection probe silanized by octadecane through hydrophobic effect, and further fluorescence quantification is carried out on the ganglioside by using in vitro hybridization chain reaction.

Description

Buoyancy microsphere probe for quantitative screening of ganglioside and preparation method thereof

One, the technical field

The invention relates to a pair of buoyancy microsphere probes for quantitatively screening cell surface ganglioside and a preparation method thereof.

Second, background Art

Gangliosides are glycosphingolipids comprising one or more sialic acid terminal oligosaccharide chains. They are embedded in the cell membrane via the alkane chains of the ceramide portion, regulate cellular structure, cell adhesion and signal transduction, and serve as receptor sites for microbial toxins, bacteria and viruses. Gangliosides on the cell surface are continuously synthesized and degraded by corresponding highly specific enzymes in a way that increases or decreases the sugar units. Its aberrant glycosylation biological pathways are associated with diseases such as genetic diseases and malignant transformation of cancer. Abnormal overexpression of gangliosides of different subclasses is also strongly associated with the type and progression of tumors. Therefore, screening for gangliosides on the cell surface is of great importance to reveal their associated biological processes and potential tumor progression.

In order to realize quantitative detection of cell surface ganglioside, the patent prepares a pair of functionalized buoyancy microsphere probes, and realizes nondestructive quantitative screening of cell surface ganglioside.

Third, the invention

The purpose of the invention is: based on boric acid mediated covalent recognition of cell surface sialic acid and octadecyl molecule mediated hydrophobic interaction, extraction probes containing nucleic acid cleaving enzyme sites and boric acid terminals and collection probes containing octadecyl molecules were designed, respectively. The sialic acid containing biomolecules on the cell surface are non-destructively extracted using an extraction probe and the extract is released into solution by a nucleic acid cleaving enzyme. Then collecting lipid substance in the release, namely ganglioside, by using a collecting probe, and carrying out hybridization chain reaction through the nucleic acid fragment combined on the ganglioside to obtain a fluorescent signal for quantification. The MCF-7 breast cancer cell line is taken as a model, and the prepared probe realizes the quantification of the cell surface ganglioside.

The buoyant microsphere probe capable of quantitatively screening cell surface ganglioside provided by the invention is shown in figure 1. Using hydroxylated silica buoyancy microspheres as carriers, gradually carrying out amino silanization on the surfaces of the silica buoyancy microspheres, assembling a succinimide and maleimide double-modified polyethylene glycol connector, and further modifying functional DNA molecules with aminophenylboronic acid terminals and endonuclease sites to obtain extraction probes; and (3) directly carrying out octadecylsilylating on the hydroxylated silicon dioxide buoyancy microsphere to obtain the collecting probe.

The invention is realized by the following technical scheme:

1) the DNA molecule containing the endonuclease site is formed by hybridization of DNA1 and DNA 2. The 5' end of the DNA1 is modified with carboxyl for covalent coupling with aminophenylboronic acid; the 3' end is modified with sulfydryl and is used for being covalently connected to a maleamide group on the buoyant microsphere.

2) The extraction probe takes hydroxylated buoyancy microspheres as a carrier, amino silanization is firstly carried out, then a maleamide group is covalently modified, and finally DNA molecules containing endonuclease sites are covalently assembled; the collection probes were obtained by direct octadecylsilylation of hydroxylated buoyant microspheres as shown in figure 1.

The working principle of the invention is as follows:

the working principle of the invention is shown in figure 2: the extraction probes were first incubated with the cells on a vertical rotator and centrifuged intermittently. After several cycles, the extraction probes were separated from the cells by buoyancy under centrifugation. The extraction probe is then incubated with an endonuclease to cleave the extracted sialic acid group-containing biomolecule, along with the bound nucleic acid fragments, into solution. After removal of the extraction probe by buoyancy, a collection probe was added to the purge solution. After incubating for a period of time on a vertical rotation instrument, separating and collecting probes through buoyancy; and adding the initiation DNA of the hybrid chain reaction and a pair of hairpin DNAs into the collected probe dispersion liquid to perform hybrid chain reaction to obtain a fluorescent signal. And (3) obtaining a standard curve by using a standard ganglioside solution, and quantitatively detecting the cell surface ganglioside.

Compared with the prior art, the invention has the following characteristics:

the size of the buoyancy microsphere used in the invention is close to that of cells, and the buoyancy microsphere has the characteristics of rapid buoyancy separation capability and easy modification. The prepared extraction probe can efficiently and quickly act and separate with cells, and realizes the non-destructive extraction of the cell surface ganglioside. The prepared collecting probe can specifically collect the released ganglioside through hydrophobic interaction, and further obtains a sensitive fluorescent signal by utilizing hybridization chain reaction, thereby realizing the quantitative detection of the cell surface ganglioside.

Compared with the existing ganglioside detection method, the method has the following advantages:

1. the buoyancy microsphere probe is simple and convenient in preparation method and has high specific extraction capacity and collection capacity. The extraction process does not require cell disruption and other pretreatment steps, and is advantageous over prior methods.

2. The DNA functional molecule can efficiently release the captured biomolecule containing sialic acid group under the action of endonuclease. The nucleic acid fragments bound to gangliosides can trigger hybridization chain reactions, resulting in signal amplification.

Description of the drawings

FIG. 1 is a schematic diagram of the structure and synthesis of two buoyancy microsphere probes

FIG. 2 is a schematic diagram of the detection of cell surface ganglioside by two buoyant microsphere probes

Fifth, detailed description of the invention

Example 1: combining with figure 1, synthesizing buoyancy microsphere probe capable of quantitatively screening cell surface ganglioside

Disperse in hybridization buffer (TE +100mM NaCl and 10mM MgCl)₂pH 8.0) of 100. mu.L of DNA1 (10. mu.M) and 100. mu.L of DNA2 (10. mu.M) were incubated at 37 ℃ with mixingAnd (4) at night. The resulting hybridization product was centrifuged with a 10Kd ultrafiltration tube and dispersed in phosphate buffer (PBS, pH 7.4). Then, 20. mu.L of aminobenzeneboronic acid (APBA, 500. mu.M) and 20. mu.L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 500. mu.M) were added thereto, respectively, and stirred at room temperature overnight. The resulting functional DNA molecule (HS-DNA1/2-APBA) was centrifuged with a 10Kd ultrafiltration centrifuge tube and dispersed in TE buffer for further use.

Silica buoyant microspheres (about 2.5X 10)⁷One) was first stirred overnight with piranha (30% hydrogen peroxide: 98% sulfuric acid 1: 3) on a vertical rotary stirrer. The obtained hydroxylated buoyancy microspheres are washed and dried, and then stirred overnight with 1mL of 5% amino silanization reagent dissolved in methanol. The obtained amino silanized buoyant microspheres were washed twice with methanol and Dimethylformamide (DMF), dispersed into a DMF solution containing succinimide and maleimide di-modified polyethylene glycol (NHS-PEG-Mal, 100mM) and N, N-diisopropylethylamine (DIPEA, 150mM), stirred overnight at room temperature, and then washed twice with DMF and water. And finally, adding 100 mu L of 1 mu M HS-DNA1/2-APBA into the obtained buoyancy microsphere modified with maleimide groups, stirring overnight at room temperature, and washing twice with water to obtain the extraction probe.

Hydroxylated silica buoyant microspheres (about 1.5X 10)⁶One) was added to a methanol solution containing 5% octadecylsilane reagent, stirred overnight at room temperature, and washed twice with methanol and PBS to obtain a collection probe.

Example 2: with reference to FIG. 2, quantitative determination of gangliosides on cell surface was performed using a buoyant microsphere probe

MCF-7 cells (50. mu.L, 1.2X 10) were used as a basic model for MCF-7 breast cancer cell lines⁵mL^-1) And extraction probe (50. mu.L, 9.6X 10)⁵mL^-1) After mixing and shaking in PBS solution for 55 minutes, it was centrifuged at 1000rpm for 5 minutes. After 4 cycles, centrifugation was performed, and the floated extraction probe was taken out by buoyancy, washed twice with PBS, and added to 100. mu.L of an enzyme digestion buffer containing 2% Eco47 III. Stirring, incubating for 15 min, centrifuging, removing floating extraction probe by buoyancy, adding into the lower solution10 μ L of collection probe (9X 10)⁴mL^-1) After incubation for two hours at room temperature with stirring, the cells were centrifuged and washed twice with PBS. The resulting collected ganglioside-collected collection probe was added to 100. mu.L of hybridization buffer containing hybridization chain reaction primer (1. mu.M), hybridization chain reaction hairpin H1 (10. mu.M) and H2 (10. mu.M), and mixed and incubated at 37 ℃ for 4 hours. The resulting collection probe was centrifuged, washed twice with TE, and fluorescence imaged to obtain a quantitative fluorescent signal.

Standard solutions of ganglioside GD1a at different concentrations (50. mu.L, 0.5, 1.0, 1.5, 2.0, 2.5nM) were mixed with extraction probes (50. mu.L, 9.6X 10, respectively)⁵mL^-1) And (3) incubating in a PBS solution, performing enzyme digestion, collection and hybridization chain reaction by using the same method to obtain a standard curve, and combining a fluorescence signal of a cell experiment to obtain a quantitative detection result of the cell surface ganglioside.

Claims

1. A buoyancy microsphere probe for quantitatively screening cell surface ganglioside is characterized by comprising an extraction probe and a collection probe; the extraction probe takes silicon dioxide buoyancy microspheres as a carrier and respectively loads functional DNA molecules with endonuclease Eco47III cutting sites and boric acid terminals; the functional DNA molecule is formed by hybridizing DNA1 and DNA2, the 3' end of the DNA1 is sulfydryl, and the DNA molecule is connected to the carbon dioxide buoyancy microsphere subjected to amino silanization through a succinimide and maleimide double-modified polyethylene glycol chain; the 5' end of the DNA1 is carboxyl, and is used for specifically identifying and extracting biomolecules containing sialic acid groups on the cell surface by covalently bonding aminophenylboronic acid molecules; the DNA2 is hybridized with the middle part of the DNA1 to form a cutting site of an endonuclease Eco47 III; the extraction probe can rapidly act on and separate from cells, so that the non-destructive extraction of the cell surface ganglioside is realized; the collecting probe is obtained by directly performing octadecyl silanization on the hydroxylated silicon dioxide buoyancy microsphere, the collecting probe performs specific collection on the released ganglioside through hydrophobic effect, and a fluorescence signal is further obtained by utilizing a hybrid chain reaction, so that quantitative detection of the cell surface ganglioside is realized.

2. The buoyant microsphere probe of claim 1, wherein the collected sialic acid group containing biomolecules on the surface of the extraction probe are cleaved with the endonuclease Eco47III and released into solution along with the bound nucleic acid fragments, and the ganglioside containing molecules in the release are collected by the collection probe.

3. The buoyant microsphere probe of claim 1, wherein the ganglioside-containing molecules collected on the surface of the collection probe can hybridize with the priming sequence via the nucleic acid fragments bound thereto, and initiate a hybridization chain reaction to obtain a fluorescent signal.