CN113856778A - Microfluidic chip, device and method for selectively detecting microorganisms - Google Patents

Microfluidic chip, device and method for selectively detecting microorganisms Download PDF

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CN113856778A
CN113856778A CN202111296312.8A CN202111296312A CN113856778A CN 113856778 A CN113856778 A CN 113856778A CN 202111296312 A CN202111296312 A CN 202111296312A CN 113856778 A CN113856778 A CN 113856778A
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林金明
李楠
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Tsinghua University
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Abstract

The invention relates to a microfluidic chip, a device and a method for selectively detecting microorganisms. The microfluidic chip comprises a sample injection hole layer, a detection hole, a channel layer and a substrate layer which are sequentially arranged from top to bottom; a plurality of channels which are radially arranged are arranged on the detection holes and the channel layer, and the tail ends of the channels are provided with the detection holes; the detection hole is pre-embedded with freeze-dried powder of a reagent for selectively detecting microorganisms. The micro-fluidic chip provided by the invention is provided with a plurality of detection holes aiming at different kinds of target microorganisms, and can be used for simultaneously detecting different microorganisms. The device and the method containing the microfluidic chip can realize rapid and selective detection of a plurality of specific microorganisms, can greatly simplify the operation steps, and have portability.

Description

Microfluidic chip, device and method for selectively detecting microorganisms
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a microfluidic chip, a device and a method for selectively detecting microorganisms.
Background
Infectious diseases caused by microbial infections pose significant risks to global food safety and public health, and have become a major concern worldwide. Timely monitoring and control of microbial contamination is an important task in the food hygiene industry.
Adenosine Triphosphate (ATP) -based bioluminescent methods have been widely used in the food industry and in environmental inspections for monitoring and assessing the cleanliness of various environmental surfaces. ATP is a molecule found in living cells that produces a bioluminescent signal in the presence of luciferase, luciferin, magnesium ions and oxygen, and the luminescent signal produced is proportional to the amount of ATP released by the lysed bacteria. Generally, the amount of ATP contained in each bacterium is relatively constant, and the luminescent signal generated is therefore proportional to the concentration of bacteria in the sample.
Compared with the traditional method for detecting the microorganisms based on the culture method, the method for detecting the microorganisms based on the ATP bioluminescence method has the advantages of simplicity, rapidness and the like. However, this method has limited application in the selective detection and quantification of specific bacteria, since it does not distinguish between different species of bacteria, nor does it distinguish between ATP being measured from beneficial or harmful bacteria. Therefore, the current methods for measuring ATP levels do not provide detailed information for identifying target bacteria, and the microbial risk cannot be correctly assessed.
The micro-fluidic chip has the characteristics of small volume, small reagent dosage, simple and convenient operation and the like, and has attracted extensive attention in the field of rapid detection in recent years. In addition, the high flux of the microfluidic chip is beneficial to realizing the simultaneous detection of various microorganisms in a sample, and the detection cost can be greatly reduced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a micro-fluidic chip, a device and a method for selectively detecting microorganisms on the basis of an ATP bioluminescence detection principle. The device and the method of the microfluidic chip containing the selective detection microorganisms can realize the selective detection of specific microorganisms and can also realize the differentiation of ATP in a sample matrix to be detected and ATP in the microorganisms.
To this end, the present invention provides a microfluidic chip for selectively detecting microorganisms, which includes a sample injection hole layer, a detection hole and a channel layer, and a substrate layer, which are sequentially arranged from top to bottom;
a plurality of channels which are radially arranged are arranged on the detection holes and the channel layer, and the tail ends of the channels are provided with the detection holes; freeze-dried powder of a reagent for selectively detecting microorganisms is pre-embedded in the detection hole;
and the sample adding hole layer is provided with sample adding holes, and the sample adding holes are used for injecting samples to be detected into the detection holes and the channels on the channel layer through the sample adding holes and further enter the detection holes.
In the invention, the detection holes are arranged at the tail end of the channel, so that the number of the detection holes is the same as that of the channel.
In some embodiments of the present invention, the number of the detection holes is multiple, so that simultaneous determination of multiple microorganisms in a sample to be detected can be realized.
In some embodiments of the present invention, the number of the detection holes is 3 or more; more preferably, the number of the detection holes is 4-11.
In some embodiments of the invention, the test wells comprise one positive control well, one negative control well, and several (e.g., 1-9) test wells for different types of target microorganisms.
The number of detection holes is not limited explicitly, and the number of detection holes can be 11 or even more. In some embodiments of the present invention, the number of the detection wells is 11, wherein 1, 2, 3, 4, 6, 7, 8, and 9 wells are experimental wells, and the lyophilized photothermal conversion nanomaterials (e.g., gold nanorods) coated with different kinds of antibodies and ATP bioluminescence substrates are pre-embedded in the experimental wells, respectively, so as to detect different target microorganisms; 10 holes are used as negative control holes, and photo-thermal conversion nano materials (such as gold nanorods) coated by antibodies and ATP bioluminescence substrates are pre-buried; and 11 holes are positive control holes, and bacteria lysis reagent and ATP bioluminescence substrate are pre-buried.
In some embodiments of the invention, the reagent is a mixture comprising an antibody-coated photothermal conversion nanomaterial and an ATP bioluminescent substrate, and the antibody is capable of specifically binding to the target microorganism to be detected.
In some preferred embodiments of the present invention, the mixed solution contains the antibody-coated photothermal conversion nanomaterial at a mass concentration of 0.025-0.1 mg/mL and the ATP bioluminescent substrate at a mass concentration of 3-5 mg/mL.
In some embodiments of the invention, the mixed solution may have a mass concentration of the antibody-coated photothermal conversion nanomaterial of 0.025mg/mL and the ATP bioluminescent substrate of 5 mg/mL.
In the present invention, the preparation method of the mixed solution may be: 0.01mg of the antibody-coated photothermal conversion nanomaterial was mixed with an ATP bioluminescent substrate (2 mg of mixed substrate powder of luciferase, luciferin, and magnesium ions in total), and dissolved in 400. mu.L of a buffer solution to obtain a mixed solution containing the antibody-coated photothermal conversion nanomaterial and the ATP bioluminescent substrate, wherein the mass concentration of the antibody-coated photothermal conversion nanomaterial in the mixed solution was 0.025mg/mL, and the mass concentration of the ATP bioluminescent substrate was 5 mg/mL. The antibody type on the photothermal conversion nano material is selected according to the requirement to be detected.
In some embodiments of the invention, the photothermal conversion nanomaterial is a gold nanorod; preferably, the length-diameter ratio of the gold nanorods is 4-4.3, such as 4.1.
In some embodiments of the invention, the ATP bioluminescent substrate comprises a thermostable luciferase, luciferin and magnesium ions.
The relationship between the amounts of thermostable luciferase, luciferin and magnesium ions in the ATP bioluminescent substrate is not specifically limited in the present invention, and one skilled in the art can routinely select them as desired.
In some embodiments of the present invention, the diameter of the detection hole is 3 to 5mm, and the depth is 1 to 2 mm.
In some embodiments of the invention, the detection hole has a diameter of 5mm and a depth of 2 mm.
In other embodiments of the present invention, the width of the channel is 0.5 to 1mm, and the depth is 50 to 100 μm.
In some embodiments of the invention, the channels have a width of 1mm and a depth of 100 μm.
In some embodiments of the invention, the diameter of the sample application hole on the sample application hole layer is 2-3 mm.
In some embodiments of the invention, the sample application well on the sample application well layer has a diameter of 3 mm.
In some preferred embodiments of the present invention, the sample orifice layer is provided with vent valve wells at positions corresponding to the detection wells on the detection wells and the channel layer. The vent valve pores are used for maintaining the balance of the air pressure inside and outside the chip.
In other preferred embodiments of the present invention, the diameter of the vent valve orifice is 0.5 to 1 mm. In some embodiments of the invention, the vent valve orifice has a diameter of 1 mm.
In some embodiments of the invention, the sample orifice layer, the detection orifice and channel layer, and the substrate layer are all transparent materials. In some embodiments of the present invention, the material of the sample hole layer, the detection hole and the channel layer is polydimethylsiloxane, and the material of the substrate layer is glass.
In other embodiments of the invention, the sample orifice layer, the detection orifice and channel layers, and the substrate layer are bonded together by plasma bonding.
That is, the sample loading hole layer is fixed with the detection hole and the channel layer through plasma bonding, the sample loading hole layer serves as a top cover of the detection hole, the substrate layer is fixed with the detection hole and the channel layer through plasma bonding, and the substrate layer serves as a bottom cover of the detection hole.
In the invention, a plurality of detection holes aiming at different types of target microorganisms are arranged on the microfluidic chip, the photo-thermal conversion nano material coated with the antibody in the reagent pre-buried on the detection holes can be specifically combined on the specific microorganism, under the irradiation of a near-infrared light source, the nano material on the surface of the microorganism converts light into heat, and the microorganism is cracked, thereby releasing ATP. The released ATP can generate bioluminescence with luciferase, luciferin and magnesium ions in the reagent in the presence of oxygen, and the signal is captured by the optical detection element. Therefore, the microfluidic chip can be used for simultaneously detecting different microorganisms.
In order to prevent the reagents pre-buried in the microfluidic chip from being denatured or inactivated, the constructed microfluidic chip needs to be hermetically packaged, and the microfluidic chip needs to be unpacked when in use.
The invention provides a device for selectively detecting microorganisms, which comprises a light-shading box, a light-shading box and a detection unit, wherein the light-shading box is a hollow cuboid composed of a box body and a box cover, and the interior of the light-shading box is divided into two areas; a near-infrared light source is arranged in one region, a bracket of a detection chip is arranged above the near-infrared light source, the microfluidic chip of the first aspect is loaded on the bracket, and an optical detection element is arranged above the microfluidic chip, so that each detection hole of the microfluidic chip coaxially corresponds to one near-infrared light source and one optical detection element respectively; the other area is provided with a signal conversion element which is connected with the optical detection element;
and a display screen is arranged on the box cover of the light shielding box and connected with the signal conversion element. The display screen may directly display the detected signal values.
In some embodiments of the present invention, a chip socket is disposed on the box body on the side of the light-shielding box, and the microfluidic chip is inserted into or extracted from the chip socket.
In some embodiments of the invention, the near-infrared light source is an LED light source or a laser diode. Each near-infrared light source is coaxially corresponding to each detection hole of the micro-fluidic chip.
In other embodiments of the present invention, the optical detection element is a photodiode. Each optical detection element is coaxially corresponding to each detection hole of the microfluidic chip.
In some embodiments of the present invention, the signal conversion element includes a transimpedance amplifier, a signal acquisition card and a single chip microcomputer connected in series in sequence. The transimpedance amplifier is connected with the optical detection element and is used for converting an optical signal into a voltage signal. The voltage signal is further converted into an instrument readable numerical value through a signal acquisition card and a single chip microcomputer which are connected with the transimpedance amplifier, and the instrument readable numerical value is displayed on a display screen on the box cover.
In the invention, the display screen is also called as a data display and is used for displaying the detected signal value, so that direct reading is facilitated.
In the present invention, the device further comprises a power supply for supplying power to the device.
In a third aspect, the present invention provides a method for selectively detecting microorganisms using a device according to the second aspect of the present invention, comprising the steps of:
s1, adding a sample to be detected containing microorganisms into a sample adding hole of the microfluidic chip;
s2, opening the optical detection element, and recording the first detection signal value of each detection hole;
s3, after the microfluidic chip is incubated, the near-infrared light source is turned on to irradiate each detection hole for a period of time, the optical detection element is turned on again, and the second detection signal value of each detection hole is recorded;
and S4, calculating the difference value between the second detection signal value and the first detection signal value of each detection hole, and judging whether the sample to be detected contains the target microorganism and/or calculating the concentration of the target microorganism according to the difference value.
In step S1, the sample to be tested enters the detection hole and each channel on the channel layer through the sample addition hole, and further enters each detection hole.
In the invention, the first detection signal value is ATP in a sample matrix to be detected and is irrelevant to microorganisms; the second detection signal value is the sum of ATP released by microorganisms in the sample to be detected and ATP existing in the matrix of the sample to be detected; and the difference value of the second detection signal value and the first detection signal value is ATP released by the microorganisms in the sample to be detected.
In the invention, when a sample to be detected contains target microorganisms, the target microorganisms can be combined with the antibody coated on the photothermal conversion nano material in the reagent through incubation, so that the photothermal conversion nano material coated by the antibody is attached to the surface of the microorganisms, and then the photothermal conversion nano material attached to the surface of the microorganisms is irradiated by a near infrared light source to convert light energy into heat energy, so that the surface temperature of the microorganisms is increased, and the microorganisms are thermally cracked to release ATP.
In some embodiments of the present invention, when the difference between the second detection signal value and the first detection signal value of the detection well is greater than 0, it indicates that the target microorganism detected by the detection well is contained in the sample to be tested.
In other embodiments of the present invention, when the difference between the second detection signal value and the first detection signal value of the detection well is greater than 0, the difference of the detection well is brought into a standard curve of the concentration of the target microorganism detected by the detection well and the difference of the detection signal, and the concentration of the target microorganism is calculated.
In the present invention, the standard curve of the difference between the concentration of the target microorganism and the detection signal can be obtained by detecting a series of target microorganism samples with known concentrations.
In the present invention, the detection signal difference refers to the difference between the second detection signal value and the first detection signal value of a target microorganism sample of known concentration.
In some embodiments of the present invention, in step S3, the incubation time is 15 to 20 minutes (for example, 20 minutes), and the incubation temperature is 15 to 37 ℃.
In other embodiments of the present invention, in step S3, the irradiation time is 5 to 10 minutes, for example, 6 minutes.
In some embodiments of the invention, the method is specifically operated as follows: and adding a sample to be detected containing microorganisms into the sample adding holes of the microfluidic chip, wherein the sample to be detected enters the channel of the middle layer from the sample adding hole on the upper layer and then enters each detection hole. Immediately, the optical detection element is turned on and the first detection signal value is recorded. This signal value is the presence of ATP in the matrix of the sample to be tested, independent of the microorganisms, i.e.the background signal. And (3) incubating for 15-20 minutes at 15-37 ℃, and binding the antibody-coated photothermal conversion nano material (such as gold nanorods) to the surface of the target microorganism. And (3) opening a near-infrared light source to irradiate the detection hole for 5-10 minutes, and then closing the detection hole, wherein the photo-thermal conversion nano material (such as a gold nanorod) attached to the surface of the microorganism converts light energy into heat energy, so that the surface temperature of the microorganism is raised until the ATP is released by the thermal cracking of the microorganism. The optical detection element is turned on again and the second detection signal value is recorded. The signal value is the sum of ATP released by the microorganisms in the sample to be detected and ATP in the matrix of the sample to be detected, and the difference value of the two is ATP released by the microorganisms in the sample to be detected. Then, the difference can be used to judge whether the sample to be tested contains the target microorganism and/or calculate the concentration of the target microorganism.
The invention has the beneficial effects that: the micro-fluidic chip provided by the invention is provided with a plurality of detection holes aiming at different kinds of target microorganisms, and can be used for simultaneously detecting different microorganisms. The device and the method containing the microfluidic chip can realize rapid and selective detection of a plurality of specific microorganisms, can greatly simplify the operation steps, and have portability.
Drawings
The invention will be further explained with reference to the drawings.
FIG. 1 is a schematic diagram of the structure of a microfluidic detection chip used in example 1; wherein the reference numerals have the following meanings: 101-microfluidic detection chip; 1-a layer of sample-orifice; 2-detection holes and channel layers; 3-a base layer; 4-a sample application hole; 5-vent valve orifice; 6-channel; 7-a first detection well; 8-a second detection well; 9-a third detection well; 10-a fourth detection well; 11-a fifth detection well; 12-a sixth detection well; 13-a seventh detection well; 14-eighth detection well; 15-ninth detection well; 16-tenth detection well; 17-eleventh detection well.
Fig. 2 is an external view of the microfluidic detection chip used in example 1.
FIG. 3 is a view showing the construction of the exterior of the apparatus for selectively detecting microorganisms employed in example 1; wherein the reference numerals have the following meanings: 24-a display screen; 26-chip socket.
FIG. 4 is a view showing the construction of the inside of the apparatus for selectively detecting microorganisms employed in example 1; wherein the reference numerals have the following meanings: 18-an LED light source; 19-a scaffold; 20-a photodiode; 21-a transimpedance amplifier; 22-signal acquisition card; 23-a single chip microcomputer; 24-a display screen; 25-a power supply; 101-microfluidic detection chip.
FIG. 5 is a graph showing the standard curve of the difference between the concentration and the detection signal of the microorganisms corresponding to Escherichia coli, Staphylococcus aureus, Salmonella and Bacillus cereus in example 1.
Detailed Description
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
A schematic structural diagram and an appearance diagram of a microfluidic chip 101 for selectively detecting microorganisms used in the following example 1 are respectively shown in fig. 1 and 2, and the microfluidic chip includes a sample injection hole layer 1, a detection hole and channel layer 2, and a substrate layer 3, which are sequentially arranged from top to bottom; the sample injection hole layer 1, the detection hole and channel layer 2 and the substrate layer 3 are bonded together through plasma bonding;
the detection holes and the channel layer 2 are provided with 11 channels 6 which are radially arranged, and the tail ends of the 11 channels are provided with detection holes which are respectively a first detection hole 7, a second detection hole 8, a third detection hole 9, a fourth detection hole 10, a fifth detection hole 11, a sixth detection hole 12, a seventh detection hole 13, an eighth detection hole 14, a ninth detection hole 15, a tenth detection hole 16 and an eleventh detection hole 17; freeze-dried powder of a reagent for selectively detecting microorganisms is pre-embedded in the detection hole;
the sample adding hole layer 1 is provided with sample adding holes 4, and the sample adding holes 4 are used for injecting a sample to be detected into the detection holes and each channel 6 on the channel layer 2 through the sample adding holes 4 and further entering each detection hole; a vent valve small hole 5 is also arranged on the sample injection hole layer 1 corresponding to the detection hole on the detection hole and the channel layer 2;
the diameter of the detection hole is 5mm, the depth of the detection hole is 2mm, the width of the channel is 1mm, and the depth of the channel is 100 micrometers; the diameter of the sample adding hole is 2mm, and the diameter of the small hole of the vent valve is 1 mm.
The external structural view and the internal structural view of the apparatus for selectively detecting microorganisms employed in example 1 described below are shown in FIGS. 3 and 4, respectively; the light-shading box comprises a light-shading box, wherein the light-shading box is a hollow cuboid composed of a box body and a box cover, and the interior of the light-shading box is divided into two areas; an LED light source 18 is arranged in one area, a bracket 19 of a detection chip is arranged above the LED light source 18, a microfluidic chip 101 is loaded on the bracket 19, and a photodiode 20 is arranged above the microfluidic chip 101, so that each detection hole of the microfluidic chip 101 coaxially corresponds to one LED light source 18 and one photodiode 20 respectively; the other region is provided with a signal conversion element connected with the photodiode 20;
a display screen 24 is mounted on a box cover of the light shielding box, and the display screen 24 is connected with the signal conversion element;
a chip socket 25 is arranged on the box body on the side surface of the light shielding box, and the microfluidic chip 101 is inserted into or pulled out of the chip socket 25;
the signal conversion element comprises a transimpedance amplifier 21, a signal acquisition card 22 and a single chip microcomputer 23 which are sequentially connected in series; the transimpedance amplifier is connected with the photodiode 20 and is used for converting an optical signal into a voltage signal; the voltage signal is further converted into an instrument readable numerical value through a signal acquisition card 22 and a single chip microcomputer 23 which are connected with the transimpedance amplifier 21, and the instrument readable numerical value is displayed on a display screen 24 on the box cover;
the device further comprises a power supply 25, the power supply 25 being adapted to power the device.
Example 1: and rapidly and selectively detecting escherichia coli, staphylococcus aureus, salmonella and bacillus cereus in the milk sample.
The freeze-dried powder of the mixed liquid of gold nanorods coated by an escherichia coli antibody and an ATP bioluminescence substrate is pre-embedded in a first detection hole of the microfluidic detection chip, the freeze-dried powder of the mixed liquid of the gold nanorods coated by a staphylococcus aureus antibody and the ATP bioluminescence substrate is pre-embedded in a second detection hole, the freeze-dried powder of the mixed liquid of the gold nanorods coated by a salmonella antibody and the ATP bioluminescence substrate is pre-embedded in a third detection hole, the freeze-dried powder of the mixed liquid of the gold nanorods coated by a bacillus cereus antibody and the ATP bioluminescence substrate is pre-embedded in a fourth detection hole, the freeze-dried powder of the mixed liquid of the gold nanorods coated by a non-antibody and the ATP bioluminescence substrate is pre-embedded in a fifth detection hole, and the freeze-dried powder of the mixed liquid of a bacterial lysis reagent and the ATP bioluminescence substrate is pre-embedded in a sixth detection hole. The fifth detection hole is a negative control hole, and no gold nanorod is attached to the surface of the bacteria because the gold nanorod is not coated with the antibody, so that the bacteria are not cracked and ATP is released. The sixth detection hole is a positive control hole, and the bacterial lysis reagent can lyse all bacteria to release ATP. The mass concentration of the gold nanorods coated by the corresponding antibodies in the mixed solution pre-buried in the first detection hole and the fourth detection hole is 0.025mg/mL, the length-diameter ratio of the gold nanorods is 4.1, and the mass concentration of the ATP bioluminescence substrate (mixed substrate powder of luciferase, fluorescein and magnesium ions) is 5 mg/mL.
The detection steps are as follows: in the detection process, after a milk sample is injected into each detection hole from the sample injection hole, signal detection is carried out by using a photodiode immediately, and first detection signal values of 6 detection holes are recorded, wherein the first detection signal values in the first detection hole and the fourth detection hole are respectively 0.25, 0.26, 0.19 and 0.3, and the signal values at the moment are ATP in a milk substrate. The microfluidic chip was incubated at room temperature for 20 minutes to allow the antibody-coated gold nanorods to bind to the surface of the target microorganism. And then 6 LED light sources positioned at the bottom of the microfluidic chip are opened to irradiate the corresponding detection holes for 6 minutes and then are closed, so that the gold nanorods attached to the surfaces of the bacteria in the 6 different detection holes are subjected to photothermal conversion, and the bacteria are cracked to release ATP. And performing signal detection again by using the photodiode and recording second detection signal values of the 6 detection holes, wherein the second detection signal values in the first detection hole to the fourth detection hole are respectively 2.09, 1.782, 1.8 and 2.26, and the signal value at the moment is the sum of ATP released by the corresponding microorganisms in the milk sample and ATP in the substrate.
The differences between the second detection signal value and the first detection signal value (the ATP released by the corresponding specific microorganism in the milk sample) in the first detection well to the fourth detection well are calculated to be 1.84, 1.522, 1.61, and 1.96, respectively, and the differences are brought into the corresponding standard curves (as shown in fig. 5), so as to obtain the concentrations of escherichia coli, staphylococcus aureus, salmonella, and bacillus cereus in the milk sample.
The concentration of colibacillus, staphylococcus aureus, salmonella and bacillus cereus in the milk sample is respectively 10 by calculation5CFU/mL、105CFU/mL、105CFU/mL、105CFU/mL。
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims (10)

1. A micro-fluidic chip for selectively detecting microorganisms comprises a sample injection hole layer, a detection hole, a channel layer and a basal layer which are sequentially arranged from top to bottom;
a plurality of channels which are radially arranged are arranged on the detection holes and the channel layer, and the tail ends of the channels are provided with the detection holes; freeze-dried powder of a reagent for selectively detecting microorganisms is pre-embedded in the detection hole;
the sample adding hole layer is provided with sample adding holes, and the sample adding holes are used for injecting a sample to be detected into the detection holes and the channels on the channel layer through the sample adding holes and further entering the detection holes;
preferably, the number of the detection holes is more than 3; more preferably, the number of the detection holes is 4-11.
2. The microfluidic chip according to claim 1, wherein the reagent is a mixture comprising an antibody-coated photothermal conversion nanomaterial and an ATP bioluminescent substrate, and the antibody is capable of specifically binding to the target microorganism to be detected;
preferably, the mass concentration of the antibody-coated photothermal conversion nanomaterial in the mixed solution is 0.025-0.1 mg/mL, and the mass concentration of the ATP bioluminescence substrate is 3-5 mg/mL.
3. The microfluidic chip according to claim 2, wherein the photothermal conversion nanomaterial is gold nanorods; preferably, the length-diameter ratio of the gold nanorods is 4-4.3; and/or
The ATP bioluminescent substrate includes thermostable luciferase, luciferin, and magnesium ions.
4. The microfluidic chip according to any one of claims 1 to 3, wherein the detection hole has a diameter of 3 to 5mm and a depth of 1 to 2 mm; and/or
The width of the channel is 0.5-1 mm, and the depth is 50-100 μm.
5. The microfluidic chip according to any one of claims 1 to 4, wherein the diameter of the sample application hole on the sample application hole layer is 2 to 3 mm;
preferably, the sample injection hole layer is provided with a vent valve pore corresponding to the detection hole on the detection hole and the channel layer; further preferably, the diameter of the small hole of the vent valve is 0.5-1 mm.
6. The microfluidic chip according to any of claims 1 to 5, wherein the sample injection hole layer, the detection holes and channels layer, and the substrate layer are all made of transparent materials; and/or
The sample injection hole layer, the detection hole and channel layer and the substrate layer are combined together through plasma bonding.
7. A device for selectively detecting microorganisms comprises a light-shading box, wherein the light-shading box is a hollow cuboid composed of a box body and a box cover, and the interior of the light-shading box is divided into two areas; a near-infrared light source is arranged in one area, a bracket of a detection chip is arranged above the near-infrared light source, the microfluidic chip as claimed in any one of claims 1 to 6 is loaded on the bracket, and an optical detection element is arranged above the microfluidic chip, so that each detection hole of the microfluidic chip coaxially corresponds to one near-infrared light source and one optical detection element respectively; the other area is provided with a signal conversion element which is connected with the optical detection element;
a display screen is arranged on a box cover of the light shielding box and connected with the signal conversion element;
preferably, a chip socket is arranged on the box body on the side surface of the light shielding box, and the microfluidic chip is inserted into or pulled out of the chip socket.
8. A method for selectively detecting microorganisms using the device of claim 7, comprising the steps of:
s1, adding a sample to be detected containing microorganisms into a sample adding hole of the microfluidic chip;
s2, opening the optical detection element, and recording the first detection signal value of each detection hole;
s3, after the microfluidic chip is incubated, the near-infrared light source is turned on to irradiate each detection hole for a period of time, the optical detection element is turned on again, and the second detection signal value of each detection hole is recorded;
and S4, calculating the difference value between the second detection signal value and the first detection signal value of each detection hole, and judging whether the sample to be detected contains the target microorganism and/or calculating the concentration of the target microorganism according to the difference value.
9. The method of claim 8, wherein when the difference between the second detection signal value and the first detection signal value of the detection well is greater than 0, it indicates that the target microorganism detected by the detection well is contained in the sample to be tested; and/or
And when the difference value between the second detection signal value and the first detection signal value of the detection hole is larger than 0, bringing the difference value of the detection hole into a standard curve of the concentration of the target microorganism detected corresponding to the detection hole and the difference value of the detection signal, and further calculating to obtain the concentration of the target microorganism.
10. The method according to claim 8 or 9, wherein in step S3, the incubation time is 15-20 minutes, and the incubation temperature is 15-37 ℃; and/or
In step S3, the irradiation time is 5 to 10 minutes.
CN202111296312.8A 2021-11-03 2021-11-03 Microfluidic chip, device and method for selectively detecting microorganisms Pending CN113856778A (en)

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