High-flux ultra-sensitive detection dot array enhancement chip

Abstract

The disclosure discloses a high-flux and ultra-sensitive detection dot array enhancement chip, and belongs to the field of food safety detection. In the disclosure, single-layer Au nano-particles are chemically bonded onto a hydrophilic substrate, an Au nano-material is naturally deposited in holes of the chip under an electrostatic adsorption action, and a regular dot array is formed. Au particles distributed in the holes are separated with a particle surfactant (CTAB) to form plasma gaps so as to enhance the self-assemble of Au nano-particles distributed on hot-spots for a long range effect, thereby improving the sensing signal in detection efficiency and sensitivity of the chip.

Claims

1. A method of use of a dot array chip for detection of a signal, wherein the dot array chip has a dot array in a form of assembly of Au nano-particles, and particle assemble spots present a plasma enhancement effect in a form of signal enhancement micro-regional spots; wherein the method comprises placing a to-be-detected sample onto the dot array chip to detect a fluorescence intensity, wherein the dot array chip is a Au nano-particle dot array connected with a nucleotide sequence capable of capturing to-be-detected mRNA; wherein the Au nano-particle dot array is in a form of assembly of Au nano-particles, and particle assemble spots present a plasma enhancement effect in a form of signal enhancement micro-regional spots.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 shows a flow diagram of preparation of a hot-spot enhancement array chip substrate based on a microsphere etching dot array technology and a scanning electron microscope (SEM) image of holes of a chip after microsphere removal.

(2) FIG. 2A is a schematic diagram of a glass substrate plasma hot-spot enhancement array chip.

(3) FIG. 2B is a simulation diagram of nano-particle regional plasma enhancement.

(4) FIG. 3 is an inverted fluorescence microscope image (10?100) of chip holes marked with FITC-BSA.

(5) FIG. 4 is an SEM image of a nano-Au hot-spot dot array.

(6) FIG. 5A is a single-dot scanning image of Raman signals of an Au nano-rod and Au nanosphere hot-spot dot array chip.

(7) FIG. 5B is a regional Mapping scanning image with an R6G molecule as a probe of an Au nano-rod and Au nanosphere hot-spot dot array chip.

(8) FIG. 5C shows a size of plasma regions of the Au nano-rod and Au nanosphere hot-spot dot array chip.

(9) FIG. 6 shows fluorescence reactions detected by a chip prepared in Examples at various miRNA-21 concentrations, and curves from bottom to top in FIG. 6 represent miRNA-21 concentrations of 0, 0.00001, 0.00008, 0.0002, 0.0003, 0.0006, 0.0008, 0.0013, 0.001, 0.0016, 0.005, 0.1, 1, 10, 50, 100, 150, 200 and 400 fM respectively.

(10) FIG. 7 shows dependence of a fluorescence intensity to a logarithm of a miRNA-21 concentration within a linearity range of 0.0006-0.0016 fM and a linearity range of 0.1-100 fM, each datum is an average value (N=3) of three repeats, and an error bar represents a standard deviation of measurement.

DETAILED DESCRIPTION

Example 1 Preparation of Chip

(11) As shown in FIG. 1, FIG. 2A and FIG. 2B, the chip of the disclosure is prepared through the following steps:

(12) (1) cleaning a glass sheet and microspheres: the glass sheet is ultrasonically cleaned with absolute ethyl alcohol and ultrapure water for 30 min, the cleaned glass sheet is dried and then cleaned in a plasma cleaning machine for 15 min, and the cleaned glass sheet is preserved in ultrapure water;

(13) (2) distributing the microspheres at fixed points on a glass interface: 50 ?L of a microsphere solution with a concentration of 1 mol/L and a diameter of 2 ?m is sucked by a pipette to drop onto the glass sheet, when the microsphere solution is diffused to the bottom of the glass sheet, the glass sheet is inclined by 4 degrees, and rotate 180? along the bottom to the top to make part of the microsphere solution reflux to form a larger monolayer microsphere array;

(14) (3) the glass sheet is aluminized in a magnetron sputtering instrument to fix positions of the microspheres, and a thickness of an Al-film is regulated and controlled to be 50 nm by regulating an evpoaration time and an evaporation rate;

(15) (4) ultrasonically cleaning the glass sheet subjected to evaporation of the Al-film in ultrapure water to remove the microspheres on the interface so as to obtain a chip substrate of which the surface is covered with the Al-film with micro-nano holes, and taking an SEM image of the glass sheet;

(16) (5) sealing the chip processed in Step (4) in APTES vapor, and performing silanization treatment on the surface of the substrate so as to make substrate interfaces in the processed micro-nano holes carry positive charges or negative charges; and

(17) (6) making the Au nano-particles drop onto the surface of the substrate processed in Step (5) to make the Au nano-particles deposit and self-assemble in the micro-nano holes of the substrate and removing the Al-film so as to obtain the Au nano-particle assembled dot array chip with a micro-scale plasma enhancement effect.

Example 2 Detection of Raman signals of Au Nano-Rod (AuNR) and Au Nanosphere (AuNP) on Chip

(18) The synthesized Au nano-rod (AuNR) and the synthesized Au nanosphere (AuNP) are deposited in nano-holes, the Au nano-rod can self-assemble in the holes to form a signal enhancement hot-spot dot array, then 200 ?L of a hemoglobin Raman probe molecule is then added and ultrasonically shaken up, and the chip dot array is subjected to Raman signal detection after 1 h of depositing action. Detection settings are an exciting light wavelength of 532 nm and an exposure time of 0.5 s, and a dot array enhancement effect is calculated with spectrum peak intensity data. Detection results are as shown in FIG. 5A, and the results show that compared with pure hemoglobin molecules, the AuNR and AuNP dot array shows enhanced signals at both wavelengths of 600 cm.sup.?1 and 1121 cm.sup.?1, and AuNP also shows enhanced signals at a wavelength of 1600 cm.sup.?1. After calculation, an enhancement effect of the chip can reach 10.sup.2 times. As shown in FIG. 5B and FIG. 5C, it could be found that with the R6G as the Raman probe molecule, by performing regional plasma enhancement Mapping testing with 532 nm exciting light, the Au nano-particles at an assemble state presents a signal enhancement micro-regional distribution state. A plasma molecule signal enhancement region is expanded from a traditional slit nano-scale to about 0.8 ?m, and meanwhile an optimal enhancement factor is larger than 10.sup.9. It can be seen that the chip has a good plasma amplification property and a molecule signal enhancement property.

Example 3 Detection of Pathogenic Microorganism microRNA

(19) An Au nano-rod or Au nano-cross is coupled with DNA according to the following steps, where the Au nano-cross is coupled with DNA1 and the Au nano-rod is coupled with DNA2: 1. adding equal volumes of 100 uM ssDNA and 1 mM TCEP into a PCR tube to stand for 3 h out of light and activate DNA; 2. sucking 1 mL of a 0.1 M Au nano-rod or Au nano-cross; 3. sucking an equal volume of SDS-HCl (pH=3) as Step 1, and adding to the material in Step 2; 4. performing oscillating at room temperature for 12 h at 1000 rpm; and 5. performing centrifuging for 10 min at 1000 rpm, sucking supernate, and adding 0.005 M CTAB to precipitates for use.

(20) The Au nano-cross connected with the DNA1 and the Au nano-rod coupled with the DNA2 are deposited in holes of the chip prepared by the method of Example 1 according to a same molar weight, and in order to facilitate observation, a chain end of the DNA1 is modified with a cy5 fluorescence dye molecule in advance by a click reaction technology. In addition, the Au nano-rod is connected with the DNA2, part of basic groups of the DNA1 and the DNA2 are in complementary pairing, and at the moment, a cy5 fluorescence dye does not emit light and is at a quenched state. When pathogenic microorganism miRNA-21 is added, since part of basic groups of the RNA and the DNA1 are in complementary pairing, the DNA1 coupled with the Au nano-rod and RNA are hybridized, cy5 fluorescence of the DNA2 coupled with the Au nano-cross is released, the pathogenic microorganism miRNA-21 could be detected according to a fluorescence intensity of cy5. Detection results are as shown in FIG. 6, and it is shown in FIG. 7 that curves of a fluorescence intensity to a logarithm of an miRNA-21 concentration present two linear responses represented by equations (F=1830.32 logC+6349.27, R.sup.2=0.9901; and F=244.41 logC+1916.10, R.sup.2=0.9984); and a detection limit can be as low as 0.5 aM and 0.03 fM, which shows that the chip has a high-flux characteristic and an ultra-sensitive characteristic.

(21) The DNA1, DNA2 and microRNA sequences used are all purchased from Shanghai Bioengineering Co., LTD.

(22) TABLE-US-00002 DNA1: (SEQIDNO.1) 5-HS-C6-AAAAAATCAACATCAGTCTGATAAGCTA-3 DNA2: (SEQIDNO.2) 5-HS-C6-AAAAAAAAAAAAAAAATAGCTTATCAGACT-cy5-3 miRNA-21: (SEQIDNO.3) 5-UAGCUUAUCAGACUGAUGUUGA-3

Comparative Example 1

(23) The specific embodiment is the same as Example 2 except that the Au nano-material is not deposited in nano-holes, 200 ?L of Raman probe molecule hemoglobin is directly added to a solution of equal amounts of Au nano-rod (AuNR) and Au nanosphere (AuNP), and ultrasonically shaken up. Raman signals are detected, results are as shown in FIG. 8, and compared with the effect of FIG. 5A of the dot array chip, Raman signals in the solution are weakened by 10 times.