CN112345619A - Separation method of thallus in biological sample, mass spectrum identification method and drug sensitivity detection method - Google Patents
Separation method of thallus in biological sample, mass spectrum identification method and drug sensitivity detection method Download PDFInfo
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Abstract
The invention discloses a method for separating thalli from a biological sample, which adopts a micro-fluidic system; and comprises the following steps: a. adding a biological sample into a sample adding chamber in the microfluidic chip through a sample adding device, standing the microfluidic chip for a first preset time, and controlling a rotating device to drive the microfluidic chip to rotate centrifugally according to a first rotating speed, wherein the biological sample contains at least one of bacteria and fungi; b. after the microfluidic chip is centrifugally rotated, the rotating device is controlled to stop, the heavy suspension liquid is added into the sample adding chamber through the sample adding device, the rotating device is controlled to drive the microfluidic chip to carry out positive and negative alternate centrifugal rotation according to a second rotating speed, after the microfluidic chip is subjected to positive and negative alternate centrifugal rotation, the rotating device is controlled to drive the microfluidic chip to carry out centrifugal rotation according to a first rotating speed, and the second rotating speed is smaller than the first rotating speed. The invention discloses a mass spectrum identification method of thalli in a biological sample. The invention discloses a method for detecting drug sensitivity of thalli in a biological sample.
Description
Technical Field
The invention relates to the technical field of thallus detection, in particular to a separation method, a mass spectrum identification method and a drug sensitivity detection method of thallus in a biological sample.
Background
The rapid identification and drug sensitivity test of bacteria or fungi is used for determining whether antibiotics or antifungal drugs can effectively treat bacterial or fungal infectious diseases, and is the key point for clinically treating bacterial or fungal infection and guiding doctors to take drugs. At present, clinically, a enrichment flask is usually required to be put into a blood culture or cerebrospinal fluid infection based on bacterial infection for enrichment. After obtaining the sample bottle with the positive report, the streaking culture needs to be continued, and the time consumption is too long.
The current clinical methods for rapidly separating bacteria comprise differential centrifugation, filtration, potential capture and the like. Among them, the clinically standard procedure is streaked colony culture, which often requires overnight (12-24 hours) culture, and has a disadvantage of being time-consuming. Because the bacteria enrichment bottle contains metabolites of bacteria and cell impurities such as white blood cells in a blood sample, the steps of adding cell lysate after simple filtration, centrifuging, resuspending, centrifuging again, extracting and the like are often needed. However, the method usually requires manual intervention (such as steps of adding cell lysate, resuspension and the like), has multiple operation steps, and has the defects of time and labor waste.
Disclosure of Invention
Therefore, it is necessary to provide a method for separating bacteria from a biological sample, a method for identifying mass spectra, and a method for detecting drug sensitivity, which aim at the problems that the existing method for rapidly separating bacteria usually requires manual intervention, has many operation steps, and wastes time and labor.
A method for separating thallus from a biological sample adopts a microfluidic system, and the microfluidic system comprises the following steps:
the micro-fluidic chip is provided with a plurality of working areas, and each working area is provided with: the device comprises a sample adding chamber, a reaction chamber, a sample collecting chamber, a siphon valve and a waste liquid chamber; the sample adding chamber, the reaction chamber, the sample collecting chamber and the waste liquid chamber are sequentially arranged at intervals along the radial direction, the reaction chamber is respectively communicated with the sample adding chamber and the sample collecting chamber, and the reaction chamber is communicated with the waste liquid chamber through the siphon valve;
the sample adding device is used for adding a liquid sample into the sample adding chamber, and the liquid sample comprises a biological sample or a heavy suspension;
the rotary telescopic device is fixedly connected with the sample adding device;
the control device is electrically connected with the sample adding device and the rotary telescopic device respectively, and is used for controlling the sample adding device to rotate and stretch along the axial direction of the rotary telescopic device through the rotary telescopic device so as to enable the sample adding device to add the liquid sample into the microfluidic chip and also used for controlling the sample adding amount of the sample adding device added into the microfluidic chip;
the separation method comprises the following steps:
a. adding a biological sample into a sample adding chamber in the microfluidic chip through the sample adding device, standing the microfluidic chip for a first preset time, and controlling a rotating device to drive the microfluidic chip to rotate centrifugally according to a first rotating speed, wherein the biological sample contains at least one of bacteria and fungi;
b. and after the micro-fluidic chip is centrifugally rotated, controlling the rotating device to stop, adding the heavy suspension into the sample adding chamber through the sample adding device, controlling the rotating device to drive the micro-fluidic chip to carry out positive and negative alternate centrifugal rotation according to a second rotating speed, and after the micro-fluidic chip is subjected to positive and negative alternate centrifugal rotation, controlling the rotating device to drive the micro-fluidic chip to carry out centrifugal rotation according to the first rotating speed, wherein the second rotating speed is less than the first rotating speed.
In one embodiment, the biological sample is selected from saliva, blood, serum, plasma, tissue lysate, amniotic fluid, villi, vaginal secretions, feces, cerebrospinal fluid, or urine.
In one embodiment, the first rotation speed is set to allow the liquid sample in the microfluidic chip to flow into the waste chamber, and the second rotation speed is set to allow the samples in the reaction chamber and the sample collection chamber to be mixed, wherein the mixing includes mixing of the sediment and the liquid.
In one embodiment, the first rotation speed is 270 xg to 1470 xg, and the second rotation speed is 27 xg to 147 xg.
In one embodiment, the first centrifugation in the forward and reverse rotation in step b at the second rotation speed is in the same direction as the centrifugation in step a, and the centrifugation in step b at the first rotation speed is in the same direction as the centrifugation in step a.
In one embodiment, the first rotating speed is 270 Xg-1470 Xg, the second rotating speed is 27 Xg-147 Xg, in the step a, the first preset time is 20 s-40 s, and the centrifugation time of the first rotating speed is 250 s-350 s; in the step b, the single time of the forward and reverse centrifugation at the second rotating speed is 5-10 s respectively, the total number of the forward and reverse alternate centrifugation is 8-10, and the centrifugation time at the first rotating speed is 250-350 s.
In one embodiment, the sample adding device comprises:
the sample adding control console is electrically connected with the control device;
the injection main body is arranged on the sample adding console; and
and the control device controls the injection main body to add the liquid into the sample adding amount in the microfluidic chip along the injection head through the sample adding console.
In one embodiment, the rotary telescopic device comprises:
the rotating platform is electrically connected with the control device; and
the one end of flexible arm is fixed in the revolving stage, flexible arm is kept away from the one end of revolving stage with application of sample device fixed connection.
In one embodiment, the reaction chamber is funnel-shaped along a radial cross-sectional shape of the microfluidic chip.
In one embodiment, the reaction chamber is provided with a first side wall and a second side wall which are opposite to each other, and the included angle between the first side wall and the second side wall is 40-80 degrees.
A method for identifying bacteria in a biological sample by mass spectrometry, which comprises the step of the method for separating bacteria in a biological sample, and further comprises the following steps after the step b:
c. after the microfluidic chip is centrifugally rotated, controlling the rotating device to stop, adding mass spectrum lysate into the sample adding chamber through the sample adding device, and controlling the rotating device to drive the microfluidic chip to carry out positive and negative alternate centrifugal rotation according to the second rotating speed;
d. after the micro-fluidic chip is subjected to forward and reverse centrifugal rotation, the rotating device is controlled to drive the micro-fluidic chip to perform centrifugal rotation according to the first rotating speed;
e. after step d, a sample is removed from the sample collection chamber for mass spectrometric identification.
A method for detecting drug sensitivity of thallus in a biological sample, which comprises the step of separating the thallus in the biological sample by adopting the method for separating the thallus in the biological sample, and the method also comprises the step f: and (c) taking out the sample in the sample collection chamber after the step b is completed for carrying out Raman imaging drug sensitivity identification.
According to the invention, the micro-fluidic system is adopted to separate the thalli in the biological sample, and compared with the prior art, the micro-fluidic system can realize automatic separation of impurities and thalli in the biological sample. The control device of the microfluidic system controls the sample adding device to rotate and stretch along the axial direction of the rotary stretching device through the rotary stretching device, so that a liquid sample (a thallus-containing biological sample, mass spectrum lysate or resuspension) is added into the microfluidic chip by the sample adding device. And simultaneously, the control device controls the sample adding amount of the sample adding device added into the microfluidic chip. According to the invention, through the matching of the control device, the rotary expansion device and the sample adding device, the automatic addition of the liquid sample into the microfluidic chip can be realized without manual participation, and meanwhile, the separation and integration of thalli are completed in the microfluidic chip, so that the integration degree is high, and the device has the characteristics of time saving and labor saving. The method can separate the thallus from cell impurities in the biological sample, and the separated thallus can be directly used for mass spectrum detection and can also be used for Raman imaging drug sensitivity identification.
Drawings
Fig. 1 is a schematic structural diagram of a microfluidic system according to an embodiment of the present disclosure;
fig. 2 is a circuit block diagram of a microfluidic system according to an embodiment of the present disclosure;
fig. 3 is a schematic view of a partial structure of a microfluidic chip according to an embodiment of the present disclosure;
fig. 4 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present disclosure;
fig. 5 is an internal structural diagram of a computer device according to an embodiment of the present application;
FIG. 6 is an image of an isolated bacterium according to an embodiment of the present application after culturing in heavy water and a common bacteria culture for one hour;
FIG. 7 is a graphical representation of C-D bonds of isolated bacteria cultured in heavy aqueous medium containing varying concentrations of antibiotics according to one embodiment of the present application;
FIG. 8 is a statistical plot of the C-D signal intensity of bacteria at different antibiotic concentrations provided in one embodiment of the present application.
Description of reference numerals:
10. a microfluidic system; 100. a microfluidic chip; 101. a working area; 110. a sample addition chamber; 120. a reaction chamber; 121. a first side wall; 122. a second side wall; 130. a sample collection chamber; 140. a siphon valve; 150. a waste chamber; 160. a sample application hole; 170. a vent hole; 200. a sample adding device; 210. a sample adding console; 220. an injection main body; 230. an injection head; 300. rotating the telescoping device; 310. a rotating table; 320. a telescopic arm; 400. a control device; 500. and a rotating device.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning. The term "connected" and "coupled" when used in this application, unless otherwise indicated, includes both direct and indirect connections (couplings). In the description of the present application, it is to be understood that the terms "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are used only for convenience in describing the present application and for simplicity in description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, are not to be considered as limiting the present application.
In this application, unless expressly stated or limited otherwise, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through intervening media. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Referring to fig. 1 and 2, an embodiment of the present invention provides a microfluidic system 10, which can achieve rapid separation of bacteria in a sample of a vial for reporting positive blood, such as saliva, blood, serum, plasma, tissue lysate, amniotic fluid, villus, vaginal secretion, stool, cerebrospinal fluid, or urine, and has a wide application prospect in rapid diagnosis of bacterial infection diseases, diagnosis and treatment of critical patients, and the like.
The microfluidic system 10 comprises: a microfluidic chip 100, a sample adding device 200, a rotary expansion device 300 and a control device 400. The sample adding device 200 is used for adding a liquid sample into the microfluidic chip 100. The liquid sample comprises a biological sample and a resuspension. The rotary telescopic device 300 is fixedly connected with the sample adding device 200. The control device 400 is electrically connected to the sample adding device 200 and the rotary expansion device 300, respectively. The control device 400 is configured to control the sample adding device 200 to rotate and stretch along the axial direction of the rotary stretching device 300 through the rotary stretching device 300, so that the sample adding device 200 adds the liquid sample into the microfluidic chip 100. The control device 400 is further configured to control the sample adding amount of the sample adding device 200 added into the microfluidic chip 100.
In one embodiment, the microfluidic chip 100 may be a centrifugal microfluidic chip 100. In one embodiment, the material of the microfluidic chip 100 is not limited, for example, the microfluidic chip 100 may be made of one or more of polymethyl methacrylate, polypropylene, polyethylene, and the like.
It is understood that the specific structure of the sample adding device 200 is not limited as long as it has the function of adding the liquid sample into the microfluidic chip 100. In one embodiment, the sample application device 200 can be an automatic injector. The sample adding device 200 can also be formed by a common injector matched with a booster. The liquid sample can be automatically added into the microfluidic chip 100 through the sample adding device 200.
The liquid sample is a biological sample and a resuspension, and may further include other liquid samples, such as mass spectrometry lysate, according to the experimental requirements for subsequent separation of the bacteria, for example, the mass spectrometry lysate is used to lyse the separated bacteria so as to expose the proteins in the bacteria for mass spectrometry, that is, the microfluidic chip 100 may be directly converted from a biological sample into a bacteria lysate that can be directly used for mass spectrometry. Of course, the liquid sample may further include other functional reagents according to the requirement.
The biological sample can be saliva, blood, serum, plasma, tissue lysate, amniotic fluid, villi, vaginal secretion, feces, cerebrospinal fluid or urine and other positive samples suspected to contain bacteria or fungi to be detected.
The resuspension fluid can be normal biological gravity reagent such as physiological saline.
The mass spectrum lysis solution can comprise formic acid and acetonitrile, and the formic acid and the acetonitrile can be mixed and added into the microfluidic chip 100, and can also be added into the microfluidic chip 100 respectively.
It is understood that the specific structure of the rotary expansion device 300 is not limited as long as the device has the function of controlling the sample adding device 200 to rotate and expand along the axial direction of the rotary expansion device 300. In one embodiment, the rotary telescopic device 300 may be composed of a telescopic rod and a motor. Wherein, the telescopic link can be fixedly connected with the rotating shaft of the motor. That is, the motor can drive the telescopic rod to rotate along the axial direction of the telescopic rod, so that the liquid sample can be added into the microfluidic chip 100, and the intelligent degree is high.
It can be understood that the manner of fixedly connecting the rotary expansion device 300 and the sample adding device 200 is not limited, as long as the rotary expansion device 300 and the sample adding device 200 are fixed to each other. In one embodiment, the rotary expansion device 300 and the sample adding device 200 can be fixedly connected through a fixing rod. Specifically, one end of the fixing rod is fixedly connected to the rotary telescopic device 300. The other end of the fixing rod is fixedly connected with the sample adding device 200. The length of the fixing rod can be selected according to the distance between the microfluidic chip 100 and the rotary expansion device 300, and is not limited by specific numerical values.
In one embodiment, the control device 400 may be a smart device such as a computer or a mobile phone. In one embodiment, the control device 400 may also be an integrated chip with a control function. In one embodiment, when the micro-fluidic system 10 is in use, the rotary telescoping device 300 can be controlled by the control device 400 to rotate along the axial direction of the rotary telescoping device 300. And synchronously controlling the control device 400 to perform telescopic motion along the axial direction, so that the sample adding device 200 is positioned right above the microfluidic chip 100. At this time, the control device 400 can control the sample adding device 200 to add the liquid sample into the microfluidic chip 100, and at the same time, control the sample adding amount of the liquid sample added into the microfluidic chip 100. Therefore, the liquid sample can be added into the microfluidic chip 100 in the control mode without manual participation, and the automation degree is high.
In this embodiment, the control device 400 controls the sample adding device 200 to rotate and stretch along the axial direction of the rotary expansion device 300 through the rotary expansion device 300, so that the sample adding device 200 adds the liquid sample (biological sample, mass spectrometry lysate or resuspension solution, etc.) into the microfluidic chip 100. Meanwhile, the control device 400 controls the sample adding amount of the sample adding device 200 added into the microfluidic chip 100. This embodiment passes through controlling means 400 rotatory telescoping device 300 and the cooperation of application of sample device 200 can realize automatically to add in micro-fluidic chip 100 liquid sample need not artifical the participation, and the separation integration of thallus is accomplished in micro-fluidic chip 100 simultaneously, and the degree of integrating is high, has labour saving and time saving's characteristics.
In one embodiment, the sample application device 200 comprises: sample application console 210, injection main body 220, and injection head 230. The sample addition console 210 is electrically connected to the control device 400. The injection main body 220 is disposed on the sample application console 210. The injection head 230 communicates with the injection main body 220. The control device 400 controls the loading amount of the liquid sample loaded into the microfluidic chip 100 along the injection head 230 by the injection main body 220 through the loading console 210.
It is understood that the specific structure of the loading console 210 is not limited as long as it can control the loading amount of the liquid sample loaded into the microfluidic chip 100 along the injection head 230 by the injection main body 220. In one embodiment, the sample application console 210 can be a booster. The sample feeding console 210 can also be formed by matching a motor with an air cylinder. By the cooperation of the sample feeding console 210 and the control device 400, the sample feeding amount of the liquid sample fed into the microfluidic chip 100 by the injection main body 220 along the injection head 230 can be controlled.
In an embodiment, the manner of disposing the injection main body 220 on the sample-adding console 210 is not limited, as long as the sample-adding console 210 can control the sample-adding amount of the liquid sample added into the microfluidic chip 100 by the injection main body 220 along the injection head 230. In one embodiment, the injection body 220 can be disposed on the sample application console 210 by a snap fit. In one embodiment, the injection main body 220 can also be disposed on the sample application console 210 by screws. In one embodiment, the injection head 230 and the injection main body 220 may communicate with each other through a hose. In this embodiment, the control device 400 controls the injection main body 220 to add the liquid sample into the microfluidic chip 100 along the injection head 230 by the sample adding console 210, so that the sample adding amount does not need manual intervention, and the automation degree is high.
In one embodiment, the rotary telescopic device 300 comprises: a rotation stage 310 and a telescopic arm 320. The rotary table 310 is electrically connected to the control device 400. One end of the telescopic arm 320 is fixed to the rotating table 310. One end of the telescopic arm 320 far away from the rotating platform 310 is fixedly connected with the sample adding device 200. In one embodiment, the rotating table 310 may be rotated by a motor. In one embodiment, the telescopic arm 320 may be a conventional telescopic arm. For example, a hydraulically or pneumatically telescoping arm.
It should be understood that the manner of fixing one end of the telescopic arm 320 to the rotating platform 310 is not limited, as long as the telescopic arm 320 and the rotating platform 310 are fixed to each other. In one embodiment, the telescopic arm 320 and the rotating platform 310 may be fixed by bolts. In one embodiment, the telescopic arm 320 and the rotating platform 310 may be fixed by a snap. Similarly, the telescopic arm 320 far from the rotation platform 310 and the sample adding device 200 can also be fixedly connected by a buckle or a bolt.
The rotation table 310 can control the rotation of the sample adding device 200 by fixedly connecting the telescopic arm 320 and the rotation table 310 and the telescopic arm 320 and the sample adding device 200. Meanwhile, the sample adding device 200 can be controlled to stretch by controlling the stretching arm 320, so that the sample adding device 200 can add the liquid sample into the microfluidic chip 100, manual participation is avoided, and labor cost can be saved.
In one embodiment, the microfluidic system 10 further comprises: the device 500 is rotated. The rotating device 500 is electrically connected to the control device 400. The rotating device 500 is fixedly connected with the microfluidic chip 100. The rotating device 500 is used for driving the microfluidic chip 100 to rotate. In one embodiment, the rotating device 500 may be an asynchronous motor. In one embodiment, the rotating device 500 may also be a motor with forward and reverse rotation functions. The fixed connection between the rotating device 500 and the microfluidic chip 100 is as follows: the microfluidic chip 100 is fixedly connected with the rotating shaft of the rotating device 500. In this embodiment, the rotation device 500 drives the microfluidic chip 100 to rotate, so as to achieve centrifugal motion.
Referring to fig. 3 and 4, in one embodiment, the microfluidic chip 100 is provided with a plurality of working regions 101. Each working area 101 is provided with: a loading chamber 110, a reaction chamber 120, a sample collection chamber 130, a siphon valve 140, and a waste chamber 150. The loading chamber 110, the reaction chamber 120, the sample collection chamber 130, and the waste chamber 150 are sequentially spaced in a radial direction. The reaction chamber 120 is in communication with the loading chamber 110 and the sample collection chamber 130, respectively. The reaction chamber 120 communicates with the waste chamber 150 through the siphon valve 140. The sample adding device 200 is used for adding the liquid sample into the sample adding chamber 110.
In one embodiment, the number of the working areas 101 can be set according to actual requirements. For example, the number of the work areas 101 may be six. In one embodiment, the sequential radial spacing of the loading chamber 110, the reaction chamber 120, the sample collection chamber 130, and the waste chamber 150 means: the distance between the sample addition chamber 110 and the center of the microfluidic chip 100 is the smallest, the distance between the reaction chamber 120 and the center of the microfluidic chip 100 is the next, the distance between the sample collection chamber 130 and the center of the microfluidic chip 100 is the next, and the distance between the waste liquid chamber 150 and the center of the microfluidic chip 100 is the largest.
The sample application chamber 110, the reaction chamber 120 and the sample collection chamber 130 are sequentially spaced in the radial direction, so that the liquid sample in the sample application chamber 110 can sequentially flow into the reaction chamber 120 and the sample collection chamber 130 in the radial direction when the microfluidic chip 100 is operated for centrifugation. Meanwhile, the waste liquid in the reaction chamber 120 may be sucked into the waste liquid chamber 150 through the siphon valve 140. The microfluidic chip 100 with the above structure can improve the centrifugal effect of the liquid sample during reaction.
In one embodiment, the reaction chamber 120 is funnel-shaped along a radial cross-sectional shape of the microfluidic chip 100. The reaction chamber 120 is configured as a funnel shape along the radial cross-sectional shape of the microfluidic chip 100, so that when the microfluidic chip 100 is centrifuged, the liquid sample in the reaction chamber 120 can flow into the sample collection chamber 130 or flow into the waste chamber 150 through the siphon valve 140, thereby improving the centrifugation effect.
In one embodiment, the reaction chamber 120 is provided with opposing first and second sidewalls 121, 122. The included angle between the first side wall 121 and the second side wall 122 is 40-80 °. In one embodiment, the angle between the first sidewall 121 and the second sidewall 122 is set to 40 ° to 80 °, which further improves the centrifugal effect of the microfluidic chip 100 during centrifugation. Preferably, the included angle between the first side wall 121 and the second side wall 122 is set to 40 °, and the centrifugal effect is optimal. In one embodiment, the siphon valve 140 has a pipe depth of 200 μm. That is, the pipe depth of the siphon valve 140 is set to 200 μm, thereby further improving the centrifugal effect.
In one embodiment, the working area 101 further defines a sample application hole 160. The sample application hole 160 is communicated with the sample application chamber 110, and the sample application hole 160 is disposed on a side of the sample application chamber 110 away from the reaction chamber 120. The sample adding device 200 adds the liquid sample into the sample adding chamber 110 through the sample adding hole 160. In one embodiment, the sample addition hole 160 is disposed on a side of the sample addition chamber 110 away from the reaction chamber 120, that is, the sample addition hole 160 is disposed on a side of the sample addition chamber 110 close to a center of the microfluidic chip 100, so that when the microfluidic chip 100 is centrifuged, the liquid sample in the sample addition hole 160 can automatically flow into the sample addition chamber 110, and the liquid sample transfer efficiency is improved.
In one embodiment, the working area 101 further defines a plurality of vents 170. The sample addition chamber 110, the reaction chamber 120, and the waste chamber 150 are each communicated with one of the vent holes 170. Each of the vent holes 170 is disposed on one side of the sample application chamber 110, the reaction chamber 120, or the waste chamber 150 near the center of the microfluidic chip 100. That is, the sample application chamber 110 is communicated with one of the vent holes 170, and the vent hole 170 is disposed on one side of the sample application chamber 110 close to the center of the microfluidic chip 100. The reaction chamber 120 is communicated with one of the vent holes 170, and the vent hole 170 is disposed at a side of the reaction chamber 120 close to the center of the microfluidic chip 100.
The waste chamber 150 is communicated with one of the vent holes 170, and the vent hole 170 is disposed on a side of the waste chamber 150 close to the center of the microfluidic chip 100. In one embodiment, the waste chamber 150 may be connected to a plurality of the vent holes 170, so long as the plurality of vent holes 170 are disposed on a side of the waste chamber 150 close to the center of the microfluidic chip 100. In this embodiment, the sample addition chamber 110, the reaction chamber 120, and the waste liquid chamber 150 are respectively communicated with one vent hole 170, so that air in the microfluidic chip 100 can be conveniently discharged during centrifugation of the microfluidic chip 100, and the centrifugation effect can be improved.
An embodiment of the present invention further provides a method for separating bacteria from a biological sample, which is applied to the microfluidic system 10 described in any of the above embodiments, and can achieve separation of bacteria in a positive enrichment flask. The method is used for quickly separating thalli in samples such as saliva, blood, serum, plasma, tissue lysate, amniotic fluid, villus, vaginal secretion, excrement, cerebrospinal fluid or urine and the like, and has wide application prospect in the aspects of quick diagnosis, treatment and the like of infectious diseases caused by bacteria and fungi. The method for separating the thallus comprises the following steps:
a. adding a biological sample into a sample adding chamber in the microfluidic chip 100 through the sample adding device 200, standing the microfluidic chip 100 for a first preset time, and controlling a rotating device to drive the microfluidic chip 100 to rotate centrifugally according to a first rotating speed, wherein the biological sample contains at least one of bacteria and fungi.
In one embodiment, the sample adding device 200 can be controlled by the control device 400 to add the biological sample to the sample adding chamber 110 of the microfluidic chip 100. Specifically, the control device 400 can control the sample adding device 200 to add the biological sample to the sample adding chamber 110 of the microfluidic chip 100 through the rotary expansion device 300. Further, after the sample adding device 200 adds the biological sample to the current sample adding chamber 110, the control device 400 can control the rotating device 500 to rotate by an angle, so that the sample adding device 200 can add the biological sample to other sample adding chambers 110. This is circulated until the sample adding device 200 adds each sample adding chamber 110 of the microfluidic chip 100 with the biological sample.
In an embodiment, the specific structures of the control device 400, the sample adding device 200, the rotary expansion device 300, the microfluidic chip 100, and the rotating device 500 can refer to the above embodiments, and are not described herein again.
In one embodiment, biological samples can be added to the plurality of sample adding chambers 110 of the microfluidic chip 100 through the sample adding device 200, wherein each sample adding chamber is 150 μ L to 250 μ L. Then, covering a pressure-sensitive film on the surface of the microfluidic chip 100 (to prevent liquid from flowing out of the microfluidic chip 100 during centrifugation), standing the microfluidic chip 100 for a first preset time, and controlling a rotating device 500 to drive the microfluidic chip 100 to rotate centrifugally according to a first rotating speed.
The liquid in the biological sample is initially separated from the precipitate via step a. The liquid enters the waste chamber and the precipitate remains in the sample collection chamber or reaction chamber.
b. After the microfluidic chip 100 is centrifugally rotated, the rotating device is controlled to stop, the heavy suspension is added into the sample adding chamber through the sample adding device 200, the rotating device is controlled to drive the microfluidic chip 100 to carry out positive and negative alternate centrifugal rotation according to a second rotating speed, after the microfluidic chip 100 is subjected to positive and negative alternate centrifugal rotation, the rotating device is controlled to drive the microfluidic chip 100 to carry out centrifugal rotation according to the first rotating speed, and the second rotating speed is smaller than the first rotating speed.
The first rotation speed is set to allow the liquid sample in the microfluidic chip 100 to flow into the waste liquid chamber, so as to achieve separation, such as solid-liquid separation, separation of light impurities (cellular impurities) from the bacteria. The second rotational speed is set such that the samples in the reaction chamber and the sample collection chamber are mixed in order to achieve mixing, which includes mixing of the sediment and the liquid.
In one embodiment, after the microfluidic chip 100 is centrifugally rotated at the first rotation speed, the rotation device 500 may be controlled to stop by the control device 400. The pressure sensitive membrane is then opened and the resuspension fluid is loaded into the loading chamber 110 through the loading device 200. The specific operation flow is the same as the adding mode of the biological sample, and the detailed description is omitted here. The volume of the resuspension added during this procedure was sufficient.
After the resuspension solution is added into each sample adding chamber 110 of the microfluidic chip 100, the pressure-sensitive film is covered again, and the rotating device 500 is controlled to drive the microfluidic chip 100 to rotate in a forward-reverse alternate centrifugal mode according to a second rotating speed. Specifically, after the resuspension solution is added to each of the sample addition chambers 110 of the microfluidic chip 100, the rotation device 500 starts to rotate and allows the resuspension solution in each of the sample addition chambers 110 to flow into the reaction chamber 120. At this time, the rotating device 500 is continuously controlled to drive the microfluidic chip 100 to perform forward and reverse alternate centrifugal rotation according to the second rotating speed. The forward and reverse alternate centrifugation means that forward centrifugation is performed first, then reverse centrifugation, forward and reverse … … are performed, and the operations are repeated alternately. The purpose of the alternate centrifugation is to mix and resuspend the resuspension solution with the pellet.
In one embodiment, the first centrifugation in the forward and reverse rotation in step b is performed in the same direction as the centrifugation in step a, and the centrifugation in step b is performed at the first rotation speed in the same direction as the centrifugation in step a, so as to enhance the resuspension effect of the pellet.
The rotation speed in the centrifugation process is determined by various factors including the purpose of centrifugation (the purpose of uniform mixing and solid-liquid separation) and the structure of the microfluidic chip 100 (the position of each working area). According to the object of the invention, a specific rotational speed is designed. In one embodiment, the radius R from the center of the microfluidic chip to the sample application chamber 1101About 30.3mm, center to radius R of sample collection well 1302About 36.5 mm. The first rotation speed is 270 Xg-1470 Xg, and the second rotation speed is 27 Xg-147 Xg.
In one embodiment, the first rotational speed is 270 xg to 1470 xg, preferably 500 xg to 750 xg. The second rotation speed is 27 Xg to 147 Xg, preferably 50 Xg to 75 Xg. In step a, the first preset time is 20s to 40s, preferably 30 s. The centrifugation time at the first rotation speed is 250s to 350s, preferably 300 s. In the step b, the single time of the forward and reverse centrifugation at the second rotating speed is 5 s-10 s respectively, the total number of the forward and reverse alternate centrifugation is 8-10 times, and the centrifugation time at the first rotating speed is 250 s-350 s, preferably 300 s. Different embodiments are determined according to the purpose of centrifugation and the specific structure of the microfluidic chip 100.
And c, separating cell impurities and thalli in the biological sample through the step b, wherein the cell impurities enter a waste liquid chamber along with the heavy suspension liquid, and the thalli are left in a sample collection chamber.
After step b is completed, the precipitated bacteria may be removed from the sample collection chamber for storage or for subsequent experimental use, such as drug susceptibility testing. Or, after the step b is finished, the thallus is left in the microfluidic chip 100 and then is subjected to subsequent operation steps such as mass spectrometry by using the microfluidic system.
An embodiment of the present invention further provides a method for detecting drug sensitivity of bacteria in a biological sample, comprising the step of separating the bacteria in the biological sample by using the method for separating bacteria in a biological sample according to any of the embodiments described above, and further comprising the step of: and after the step b is finished, taking out the sample in the sample collection chamber for carrying out Raman imaging drug sensitivity identification.
The Raman Imaging drug sensitivity detection method can be found in the related patent with application No. 201880058493.0 filed by the inventor or in the article published by the inventor ("Rapid Determination of Antimicrobial biological reactivity by Stimulated Raman Scattering Imaging of D)2O Metabolic Incorporation in a Single Bacterium ", Meng Zhang et al, adv. Sci.2020,2001452), which will not be described herein. The sample treated by the method still has partial impurities, cannot be directly used for drug sensitivity test of the traditional method, but can be directly used for the inventor's previous drug sensitivity detection technology based on stimulated Raman imaging.
An embodiment of the present invention further provides a method for identifying bacteria in a biological sample by mass spectrometry, including the step of the method for separating bacteria in a biological sample according to any one of the above embodiments, further including the step of, after step b:
c. after the microfluidic chip 100 is centrifugally rotated, controlling the rotating device to stop, adding mass spectrum lysate into the sample adding chamber through the sample adding device 200, and controlling the rotating device to drive the microfluidic chip 100 to perform positive and negative alternate centrifugal rotation according to the second rotating speed;
d. after the micro-fluidic chip 100 is rotated in a forward and reverse centrifugal mode, controlling the rotating device to drive the micro-fluidic chip 100 to rotate in a centrifugal mode according to the first rotating speed;
e. after step d, a sample is removed from the sample collection chamber for mass spectrometric identification.
In one embodiment, after the microfluidic chip 100 is centrifugally rotated at the first rotation speed, the rotation device 500 may be controlled to stop by the control device 400. Mass spectrometry lysate is then added to the loading chamber 110 by the loading device 200. The specific operation flow is the same as the adding mode of the biological sample, and the detailed description is omitted here. After the mass spectrum lysis solution is added into each sample adding chamber 110 in the micro-fluidic chip 100, the rotating device 500 can be controlled to drive the micro-fluidic chip 100 to rotate in a forward and reverse centrifugal mode according to the second rotating speed, so that the thalli are lysed, and proteins are exposed for subsequent mass spectrum detection to identify strains of thalli in a biological sample.
For a specific mass spectrometric identification method, reference may be made to a commonly used method for mass spectrometric identification of bacteria, which is not described herein again.
In one embodiment, a computer device is provided, which may be a terminal, and its internal structure diagram may be as shown in fig. 5. The computer device includes a processor, a memory, a network interface, a display screen, and an input device connected by a system bus. Wherein the processor of the computer device is configured to provide computing and control capabilities. The memory of the computer device comprises a nonvolatile storage medium and an internal memory. The non-volatile storage medium stores an operating system and a computer program. The internal memory provides an environment for the operation of an operating system and computer programs in the non-volatile storage medium. The network interface of the computer device is used for communicating with an external terminal through a network connection. The computer program is executed by a processor to implement a bacteria isolation method. The display screen of the computer equipment can be a liquid crystal display screen or an electronic ink display screen, and the input device of the computer equipment can be a touch layer covered on the display screen, a key, a track ball or a touch pad arranged on the shell of the computer equipment, an external keyboard, a touch pad or a mouse and the like.
Those skilled in the art will appreciate that the architecture shown in fig. 5 is merely a block diagram of some of the structures associated with the disclosed aspects and is not intended to limit the computing devices to which the disclosed aspects apply, as particular computing devices may include more or less components than those shown, or may combine certain components, or have a different arrangement of components.
Referring to fig. 5, another embodiment of the present application provides a computer device, which includes a memory and a processor, wherein the memory stores a computer program, and the processor executes the computer program to implement the method for separating thallus from a biological sample or the step of further lysing in any of the above embodiments. I.e. step a to step b, or step a to step d.
An embodiment of the present application further provides a computer-readable storage medium, on which a computer program is stored, wherein the computer program, when executed by a processor, implements the method for separating bacteria from a biological sample or the step of further lysing as described in any of the above embodiments. I.e. step a to step b, or step a to step d.
According to the computer equipment and the computer-readable storage medium, through the steps a to b or the steps a to d, the thallus separation method can automatically add the liquid sample into the microfluidic chip 100 in the process of rapid separation or further cracking without manual participation, meanwhile, the separation and integration of bacteria are completed in the microfluidic chip 100, the operation steps are few, the integration degree is high, and the characteristics of time saving and labor saving are achieved.
The specific embodiment is as follows:
by adopting the microfluidic system shown in fig. 2, blood and bacteria are added into the sample adding chamber in the microfluidic chip 100 through the sample adding device 200, and after the microfluidic chip 100 is kept still for 30s, the rotating device is controlled to drive the microfluidic chip 100 to perform the first centrifugal rotation according to 735 xg and 300 s. And after the first centrifugation is finished, controlling the rotating device to stop, adding the resuspension (physiological saline) into the sample adding chamber through the sample adding device 200, and controlling the rotating device to drive the microfluidic chip 100 to perform forward and reverse alternate centrifugation rotation (second centrifugation) according to 75 Xg and 10 sx 10 times. And after the second centrifugation is finished, controlling the rotating device to stop, and controlling the rotating device to drive the microfluidic chip 100 to rotate centrifugally according to 735 Xg and 300s (third centrifugation).
And after the third centrifugation is finished, controlling the rotating device to stop, adding mass spectrum lysate (acid and acetonitrile with the volume ratio of 1: 1) into the sample adding chamber through the sample adding device 200, and controlling the rotating device to drive the microfluidic chip 100 to perform positive and negative alternate centrifugal rotation (the fourth centrifugation) according to 75 Xg and 10s X10 times. And after the fourth centrifugation is finished, controlling the rotating device to stop, and controlling the rotating device to drive the microfluidic chip 100 to rotate centrifugally according to 735 Xg and 300s (fifth centrifugation).
And taking out a part of the thalli after the third centrifugation for drug sensitivity identification directly, continuing to carry out the fourth centrifugation and the fifth centrifugation on the other part of the thalli, and taking out the thalli after the fifth centrifugation for mass spectrum identification.
The results of mass spectrometric identification after 105 trials carried out as described above are shown in table 1.
TABLE 1
Table 1 shows that compared with the conventional manual separation method, the method according to the present invention can automatically separate the bacteria in the biological sample, and improve the separation efficiency, and the bacteria separated by the separation method can meet the requirements of mass spectrometric detection of the biological sample, and the separation accuracy is not inferior to that of the conventional manual separation method.
Performing Raman spectrum drug sensitivity identification according to the following steps:
1. respectively adding the centrifuged thalli into bacterial culture solutions containing different antibiotic concentrations (similar to the antibiotic concentration gradient of the traditional drug sensitive method) and culturing for a period of time (about 1 hour);
2. centrifuging the sample, removing the supernatant, and then adding the bacteria samples at the bottom into a bacteria culture solution containing heavy water with the same antibiotic concentration for a period of time (1 hour);
3. centrifuging the sample, removing the supernatant, diluting the bottom bacterial sample with pure water, and preparing the sample (dripping the bacterial sample on a cover glass, wherein the surface of the cover glass is covered with a layer of polylysine in advance to adsorb bacteria;
4. after waiting for 10 minutes, placing the sample under a coherent Raman scattering microscope to observe bacterial signals;
5. the degree of bacteria inhibition by antibiotics with different concentrations is judged according to the carbon-deuterium (C-D) signal intensity after the bacteria metabolize the heavy water, so that a Minimal Inhibitory Concentration (MIC) value similar to that of the traditional drug sensitivity is obtained.
The image of the bacteria cultured in the heavy water and the common bacteria culture solution for 1 hour is shown in fig. 6, and it can be seen that the bacteria cultured in the common culture solution have no obvious carbon-deuterium (C-D) signal under a coherent raman scattering microscope, the bacteria cultured in the heavy water have carbon-deuterium signals, and other samples such as blood impurities have no obvious carbon-deuterium signals.
The imaging of the C-D chemical bond of the bacteria after the bacteria are cultured in the heavy water culture solution containing antibiotics with different concentrations is shown in FIG. 7, the statistics of the C-D signal intensity of the bacteria under different antibiotic concentrations is shown in FIG. 8, and it can be seen that when the antibiotic concentration is higher than a certain value, the signal intensity of the bacteria is remarkably reduced compared with the control group (0 mug/mL), therefore, by setting a threshold value, the minimum inhibitory concentration value of the bacteria with the inhibited heavy water metabolism can be obtained.
The results show that the thallus separation method can be applied to the Raman spectrum drug sensitivity identification of biological samples.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (12)
1. A method for separating thallus from a biological sample is characterized in that a micro-fluidic system is adopted, and the micro-fluidic system comprises:
the micro-fluidic chip is provided with a plurality of working areas, and each working area is provided with: the device comprises a sample adding chamber, a reaction chamber, a sample collecting chamber, a siphon valve and a waste liquid chamber; the sample adding chamber, the reaction chamber, the sample collecting chamber and the waste liquid chamber are sequentially arranged at intervals along the radial direction, the reaction chamber is respectively communicated with the sample adding chamber and the sample collecting chamber, and the reaction chamber is communicated with the waste liquid chamber through the siphon valve;
the sample adding device is used for adding a liquid sample into the sample adding chamber, and the liquid sample comprises a biological sample or a heavy suspension;
the rotary telescopic device is fixedly connected with the sample adding device;
the control device is electrically connected with the sample adding device and the rotary telescopic device respectively, and is used for controlling the sample adding device to rotate and stretch along the axial direction of the rotary telescopic device through the rotary telescopic device so as to enable the sample adding device to add the liquid sample into the microfluidic chip and also used for controlling the sample adding amount of the sample adding device added into the microfluidic chip;
the separation method comprises the following steps:
a. adding a biological sample into a sample adding chamber in the microfluidic chip through the sample adding device, standing the microfluidic chip for a first preset time, and controlling a rotating device to drive the microfluidic chip to rotate centrifugally according to a first rotating speed, wherein the biological sample contains at least one of bacteria and fungi;
b. and after the micro-fluidic chip is centrifugally rotated, controlling the rotating device to stop, adding the heavy suspension into the sample adding chamber through the sample adding device, controlling the rotating device to drive the micro-fluidic chip to carry out positive and negative alternate centrifugal rotation according to a second rotating speed, and after the micro-fluidic chip is subjected to positive and negative alternate centrifugal rotation, controlling the rotating device to drive the micro-fluidic chip to carry out centrifugal rotation according to the first rotating speed, wherein the second rotating speed is less than the first rotating speed.
2. The method according to claim 1, wherein the biological sample is selected from saliva, blood, serum, plasma, tissue lysate, amniotic fluid, villi, vaginal secretion, stool, cerebrospinal fluid and urine.
3. The method according to claim 1, wherein the first rotation speed is set to allow the liquid sample in the microfluidic chip to flow into the waste chamber, and the second rotation speed is set to allow the samples in the reaction chamber and the sample collection chamber to mix, and the mixing includes mixing of a precipitate and a liquid.
4. The method according to claim 3, wherein the first rotation speed is 270 Xg to 1470 Xg and the second rotation speed is 27 Xg to 147 Xg.
5. The method according to claim 1, wherein the first centrifugation in the alternating forward and reverse centrifugation at the second rotation speed in step b is performed in the same direction as the centrifugation in step a, and the centrifugation at the first rotation speed in step b is performed in the same direction as the centrifugation in step a.
6. The method according to claim 5, wherein the first rotation speed is 270 Xg to 1470 Xg, the second rotation speed is 27 Xg to 147 Xg, the first preset time is 20s to 40s, and the centrifugation time at the first rotation speed is 250s to 350s in step a; in the step b, the single time of the forward and reverse centrifugation at the second rotating speed is 5-10 s respectively, the total number of the forward and reverse alternate centrifugation is 8-10, and the centrifugation time at the first rotating speed is 250-350 s.
7. The method for separating bacterial cells from a biological sample according to any one of claims 1 to 6, wherein the sample application device comprises:
the sample adding control console is electrically connected with the control device;
the injection main body is arranged on the sample adding console; and
and the control device controls the injection main body to add the liquid into the sample adding amount in the microfluidic chip along the injection head through the sample adding console.
8. The method according to any one of claims 1 to 6, wherein the rotary telescopic device comprises:
the rotating platform is electrically connected with the control device; and
the one end of flexible arm is fixed in the revolving stage, flexible arm is kept away from the one end of revolving stage with application of sample device fixed connection.
9. The method according to any one of claims 1 to 6, wherein the reaction chamber has a funnel shape in a radial cross-sectional shape of the microfluidic chip.
10. The method according to claim 9, wherein the reaction chamber has a first side wall and a second side wall opposite to each other, and an angle between the first side wall and the second side wall is 40 ° to 80 °.
11. A method for mass spectrometric identification of bacterial cells in a biological sample, comprising the step of the method for separating bacterial cells in a biological sample according to any one of claims 1 to 10, further comprising, after step b:
c. after the microfluidic chip is centrifugally rotated, controlling the rotating device to stop, adding mass spectrum lysate into the sample adding chamber through the sample adding device, and controlling the rotating device to drive the microfluidic chip to carry out positive and negative alternate centrifugal rotation according to the second rotating speed;
d. after the micro-fluidic chip is subjected to forward and reverse centrifugal rotation, the rotating device is controlled to drive the micro-fluidic chip to perform centrifugal rotation according to the first rotating speed;
e. after step d, a sample is removed from the sample collection chamber for mass spectrometric identification.
12. A method for detecting drug sensitivity of bacteria in a biological sample, which comprises separating the bacteria in the biological sample by the method for separating bacteria in a biological sample according to any one of claims 1 to 10, and which further comprises the step f: and (c) taking out the sample in the sample collection chamber after the step b is completed for carrying out Raman imaging drug sensitivity identification.
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| CN210787394U (en) * | 2019-08-28 | 2020-06-19 | 广州万孚生物技术股份有限公司 | Micro-fluidic chip and in-vitro detection device comprising same |
| CN110551617A (en) * | 2019-09-03 | 2019-12-10 | 中国科学院北京基因组研究所 | Chip for separating body fluid bacteria and cells, manufacturing method and using method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118518454A (en) * | 2024-07-23 | 2024-08-20 | 天津医科大学第二医院 | Sample preprocessing device suitable for mass spectrum detects |
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