MICRO-FLUIDIC CHIP AND ITS MODIFICATION METHOD AND APPLICATION IN DETECTION OF THE QUANTITY OF FOOD BACTERIA

• US 20190323064A1
• Filed: 07/24/2018
• Published: 10/24/2019
• Est. Priority Date: 04/24/2018
• Status: Active Grant

• Current Assignee: Greentown Agricultural Testing Technology Co., Ltd.
• Original Assignee: Greentown Agricultural Testing Technology Co., Ltd.
• Inventors: HAO, XINGKAI, JIANG, YUQIAN, CAO, XUDONG, WANG, CHUANPI, ZHOU, MIN, SUN, MEINA, WANG, ZHEN
• Granted Patent: US 10,752,963 B2
• Time in Patent Office: Days
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2600/142 Toxicological screening, e.g. expression profiles which identify toxicity

Abstract

The present invention discloses a micro-fluidic chip and its modification method and application in detection of the quantity of food bacteria, comprising the following steps: changing functional group —CH₃ on the internal surface of micro-fluidic channel of micro-fluidic chip to functional group —OH through modification; Supplying amino silane reagent to the micro-fluidic channel; supplying dendritic polymer as modified by —COOH to internal surface of the micro-fluidic channel after drying; grafting primer of aminated aptamer RCA on terminal 5′ and hybrid from its padlock probe on dendrimer on internal surface of the micro-fluidic channel; wherein, the padlock probe is the complementary sequence of the aptamer of the target pathogenic bacteria to be tested; after that, supplying RCA reaction reagent to the micro-fluidic channel to make aptamer RCA generate long-chain aptamer in series. The present invention adopts two RCA reactions of varied functions in combination, and uses dendritic polymer to modify internal surface of the chip so as to improve identification and snatch of targets and signal enhancement, and realize integrated detection.

Claims

1. A micro-fluidic chip modification method, characterized in that the method comprises the following steps:

(1) changing functional group —

CH₃ on an internal surface of a micro-fluidic channel of a micro-fluidic chip to functional group —

OH through modification;

(2) supplying an amino silane reagent to the micro-fluidic channel for amino silane reaction once the reaction in Step (1) is completed;

(3) supplying a dendritic polymer as modified by —

COOH to the internal surface of the micro-fluidic channel for coupled reaction after drying;

(4) grafting a primer of an aminated aptamer RCA on terminal 5′ and

hybrids from its padlock probe on the denritic polymer on the internal surface of the micro-fluidic channel once the reaction in Step (3) is completed;

wherein, the padlock probe is the complementary sequence of the aptamer of the target pathogenic bacteria to be tested;

after that, supplying a RCA reaction reagent to the micro-fluidic channel to make aptamer RCA generate long-chain aptamer in series;

terminating RCA reaction through heating upon completion of reaction;

finally, supplying phosphate buffer to the micro-fluidic channel for cleaning.

2. The micro-fluidic modification method according to claim 1, characterized in that the dendritic polymer in Step (3) is polyethylenediamine dendritic polymer.

3. The micro-fluidic modification method according to claim 1, characterized in that Step (4) comprises the following steps:

(4-1) in a 50 μ

l reaction system, heating 2 μ

M primer of aptamer RCA and 2 μ

M padlock probe under the temperature of 95°

C. for 5 minutes before cooling down to the room temperature;

then being placed into 37°

C. water bath, and waiting for 30 min before adding 10 U T4 DNA ligase and 5 μ

l 10×

T4 DNA ligase buffer for reaction under the room temperature for 30 min;

after that, being heated to the temperature of 65°

C., and maintaining the temperature for 10 min before terminating the reaction to obtain a primer of aptamer RCA and hybrids of its padlock probe;

(4-2) once the hybrid is grafted on the internal surface of the micro-fluidic channel through carboxyl condensation reaction, supplying a RCA reagent;

in a 100 μ

l reaction system, the RCA reagent comprising 10 U phi29 DNA polymerase, 10 μ

l phi29 DNA polymerase buffer and 4 μ

l/10 mM dNTPs;

following reaction under the temperature of 37°

C. for 10 hours, maintaining the whole reaction system for 10 min under the temperature of 65°

C. before terminating the reaction.

4. The micro-fluidic chip modification method according to claim 1, characterized in that the primer of the aptamer RCA has the following sequence:

SEQ ID NO.1.

5. The micro-fluidic chip modification method according to claim 4, characterized in that padlock probe of primer of the aptamer RCA has the following sequence:

SEQ ID NO.2.

6. A micro-fluidic chip obtained through modification with the modification method according to claim 1.

7. A method of using a micro-fluidic chip in detection of the quantity of food bacteria according to claim 6.

8. The method of using the micro-fluid chip in detection of the quantity of food bacteria according to claim 7, characterized in that the method comprises the following steps:

(1) supplying a sample solution to be tested into the micro-fluidic channel;

once the internal surface of the micro-fluidic channel on the micro-fluidic chip as modified by the target pathogenic bacteria in the sample solution to be tested is identified and captured, supplying aptamer of signal amplified RCA-primer compound and padlock probe into the micro-fluidic channel, and ensuring its identification and specific adsorption on the surface of target pathogenic bacteria;

(2) putting a fluorescent probe subjecting to specific identification with signal amplified RCA into the micro-fluidic channel;

following reaction excitation, producing a binding site for fluorescent probe by signal amplified RCA;

(3) observing a reaction area of the micro-fluidic chip under the fluorescent microscope, and taking photos;

using photo processing software to convert the fluorescent photos into quantitative fluorescent signals;

comparing the detection signals with standard curve to realize quantitative detection of foodborne pathogens.

9. The method of the micro-fluid chip in detection of the quantity of food bacteria according to claim 8, characterized in that aptamer-primer compound of the signal amplified RCA has the following sequence:

SEQ ID NO.3;

the padlock probe corresponding to the aptamer-primer compound of the signal amplified RCA has the following sequence;

SEQ ID NO.4.

10. The method of the micro-fluid chip in detection of the quantity of food bacteria according to claim 8, characterized in that the fluorescent probe has the following sequence:

SEQ ID NO.5.

11. The method of the micro-fluid chip in detection of the quantity of food bacteria according to claim 9, characterized in that the fluorescent probe has the following sequence:

SEQ ID NO.5.