CN114130436A - Microfluidic chip for online pretreatment of sample and preparation method and application method thereof - Google Patents
Microfluidic chip for online pretreatment of sample and preparation method and application method thereof Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D61/36—Pervaporation; Membrane distillation; Liquid permeation
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D61/36—Pervaporation; Membrane distillation; Liquid permeation
- B01D61/366—Apparatus therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/36—Pervaporation; Membrane distillation; Liquid permeation
- B01D61/368—Accessories; Auxiliary operations
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4022—Concentrating samples by thermal techniques; Phase changes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
- B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0631—Purification arrangements, e.g. solid phase extraction [SPE]
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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- B01L2300/0887—Laminated structure
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4022—Concentrating samples by thermal techniques; Phase changes
- G01N2001/4027—Concentrating samples by thermal techniques; Phase changes evaporation leaving a concentrated sample
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
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- Clinical Laboratory Science (AREA)
- Physics & Mathematics (AREA)
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- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a microfluidic chip for sample online pretreatment and a preparation method and a using method thereof, wherein the microfluidic chip comprises an automatic extraction and filtration module, a pass-through purification module and an online pervaporation module which are sequentially communicated; the automatic extraction and filtration module is used for removing particle impurities in the sample, and the through type purification module is used for removing fat and phospholipid in the sample; the online pervaporation module is used for removing the solvent generated by the automatic extraction and filtration module. The method does not need any off-line manual operation, the whole analysis and detection time is less than 30 minutes, and the method is shortened by about 10 times compared with the traditional LC-MS/MS method; the microfluidic integrated chip prepared by the invention can be combined with platforms such as a biochip and a mass spectrometer (chip-MS), and can realize full-automatic multi-class and multi-residue analysis of various veterinary drugs.
Description
Technical Field
The invention relates to the technical field of sample pretreatment for food safety supervision or food analysis, in particular to a micro-fluidic chip for sample online pretreatment and a preparation method and a use method thereof.
Background
The microfluidic chip analysis has the advantages of high analysis speed, high integration level, automatic operation and the like, and is widely applied to the fields of clinic, biology, medicine, agriculture, food safety and the like. However, the on-line microfluidic chip capable of being coupled with the MS method reported at present does not pay attention to the simultaneous separation and extraction of various targets, because it is difficult to enrich various drugs having a wide polarity range and characteristics in one way. In cases where it is difficult to find an adsorbent to enrich for multiple drugs, another approach to achieve multi-residue detection is to remove matrix interferents such as protein or fat co-extracts, and retain the target. For matrix removal in non-chip systems, a rapid, simple, inexpensive, effective, robust, safe (QuEChERS) based strategy is widely reported for the detection of various pesticide residues in fruits and vegetables. However, the QuEChERS method still has many limitations for the detection of veterinary drug residues in animal products. For example, this operation is labor intensive and requires manual nitrogen purging to improve sensitivity. Phase separation using inorganic salts in QuEChERS results in the loss of sensitive or polar analytes. In microfluidic chip systems, Solid Phase Extraction (SPE) is the most widely used purification method based on the retention-elution principle. It is less suitable for removing fats, especially phospholipids. In fact, the main substrates removed in animal food are proteins and fats. Therefore, it is the first attempt to apply a pass-through SPE scheme by selecting an appropriate matrix to remove particles in a microfluidic system.
On the other hand, although the Pass-through SPE protocol can greatly reduce the experimental steps, it has the disadvantage of not being able to enrich the target while removing the matrix interference. To improve the sensitivity of the target, our studies considered evaporation of part of the solvent. In conventional analytical methods, most of the evaporation steps under nitrogen are performed manually in the sample preparation, which is usually the longest and more time-consuming step. The integration of such a combination pass-through module and an in-line solvent evaporation module has not been reported. The volume is reduced by pervaporation or gas and vapor transport across the membrane but the analyte is preserved to improve sensitivity. Polydimethylsiloxane (PDMS) membranes are non-porous and dense polymer membranes. Under the push of the vapor partial pressure difference of the components in the liquid mixture, the components are separated by utilizing the difference of the dissolution and diffusion speeds of the components through the membrane.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a microfluidic chip for online pretreatment of a sample; another object of the present invention is to provide a method for preparing a microfluidic chip for on-line sample pretreatment; another object of the present invention is to provide a method for using a microfluidic chip for online sample pretreatment.
The technical scheme is as follows: the invention relates to a micro-fluidic chip for online pretreatment of a sample, which comprises an automatic extraction and filtration module, a pass-through purification module and an online pervaporation module which are sequentially communicated; the automatic extraction and filtration module is used for removing particle impurities in the sample, and the straight-through purification module is used for removing fat and phospholipid in the sample; the online pervaporation module is used for removing the solvent generated by the automatic extraction and filtration module.
Further, the automatic extraction and filtration module is a micro-column array with the pitch of 100-400 μm.
Furthermore, the straight-through purification module is of a three-channel parallel structure, and enhanced matrixes are filled in the channels to remove lipid particles.
Further, each channel has a length of 3-10cm and a width of 4-10mm, and the loading of the enhanced matrix removal lipid particle per channel is 10-100 mg.
Furthermore, the online pervaporation module has a multilayer structure and comprises a liquid layer, a membrane layer and an airflow layer, wherein the liquid layer and the airflow layer have the same channel pattern and are both serpentine channel patterns.
Further, the flow direction of the liquid in the liquid layer and the gas in the gas layer is reversed.
Furthermore, the online pervaporation module is arranged on the heating plate, and inert gas is introduced into the inlet of the airflow layer.
A preparation method of the microfluidic chip comprises the following steps:
(1) etching the channel patterns of the automatic extraction and filtration module, the straight-through purification module and the online pervaporation module on a glass substrate;
(2) pouring the PDMS prepolymer into another smooth glass substrate, curing PDMS, stripping the PDMS replica from the glass mold, perforating and sealing the position corresponding to the glass substrate with the channel to obtain a chip integrating the automatic extraction and filtration module and the straight-through purification module;
(3) and (3) spin-coating PDMS on a clean glass substrate to obtain a PDMS film with a certain thickness, bonding the PDMS film with a PDMS gas layer with a serpentine channel, peeling the PDMS film and the film layer from the glass substrate, and bonding the other surface of the film layer with the serpentine channel of the glass substrate to obtain the microfluidic chip.
The application method of the microfluidic chip comprises the steps of combining the microfluidic chip with a biochip and/or a mass spectrum to determine the content of residual drugs in a sample; the sample to be detected flows into the pervaporation module through the extraction and filtration module and the pass-through purification module, and the solution is collected by the collection ring and is directly introduced into the MS detector through the switching valve or is used for biochip detection.
Further, when the sample is milk and the component to be detected is a veterinary drug, the working temperature of the online pervaporation module is 60-70 ℃, and the thickness of the film layer is 40-70 μm.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: (1) the method does not need any off-line manual operation, the whole analysis and detection time is less than 30 minutes, and the method is shortened by about 10 times compared with the traditional LC-MS/MS method;
(2) the consumption of samples and reagents is about 20 times less than that of the traditional LC-MS/MS method;
(3) the microfluidic integrated chip can be combined with platforms such as a biochip and a mass spectrometer (chip-MS) and can realize the full-automatic multi-class and multi-residue analysis of various veterinary drugs.
Drawings
FIG. 1 is a schematic diagram of a microfluidic chip for on-line sample pretreatment;
FIG. 2 is a schematic structural view of an in-line pervaporation module (M3);
FIG. 3 is a graph of the impact of the purging method and the calculation method on ME;
FIG. 4 is a graph of the effect of EMR-lipid decontamination on the intensity of targets in a matrix-matched standard solution: IMQ, (a) quantitative ionic strength with EMR-lipid decontamination, (b) quantitative ionic strength without EMR-lipid decontamination, (c) qualitative ionic strength with EMR-lipid decontamination, (d) qualitative ionic strength without EMR-lipid decontamination;
FIG. 5 is a graph of the effect of temperature and film thickness on evaporation rate;
FIG. 6 is a graph of the effect of temperature on analyte recovery;
FIG. 7 is a graph showing the effect of on-line evaporation on target signal intensity.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
Example 1
As shown in fig. 1, the microfluidic chip for online sample pretreatment comprises an automatic extraction and filtration module (M1), a pass-through purification module (M2), and an online pervaporation module (M3) which are sequentially connected; specifically, the automatic extraction and filtration module is used for removing particle impurities in the sample, and the pass-through purification module is used for removing fat and phospholipid in the sample; the online pervaporation module is used to remove the solvent generated by the automatic extraction and filtration module.
Optionally, M1 is a micro-column array with a pitch of 100-400 μ M; m2 is a three-channel parallel structure, enhanced matrix (EMR-Lipid) removal Lipid particles are filled in the channels, the length of each channel is 3-10cm, the width of each channel is 4-10mm, and the filling amount of the enhanced matrix removal Lipid particles in each channel is 10-100 mg; m3 included a liquid layer (L channel) made from a glass slide with a serpentine channel (500 μ M wide by 50 μ M deep) pattern, a membrane, and a gas flow layer (G channel) with a total channel length of 823 mm and a width of 550 μ M made from PDMS.
Alternatively, the flow direction of the liquid and gas in M3 was kept counter current rather than co-current, and a regulated inert gas source (nitrogen) was connected to the inlet of the gas flow layer, which was placed on a precision electrical heating plate, with a temperature error controlled to within + -1 deg.C.
The preparation of the microfluidic chip for online sample pretreatment comprises the following steps:
(1) preparing a glass mold:
transfer of channel pattern to glass substrate: briefly, a laser printed film having a design pattern was transferred to a spin-on chrome plate, and then subjected to ultraviolet irradiation for 10 minutes, rinsing with 0.5% (w/v) NaOH solution for 5 minutes, and soaking in a dechroming solution for 2 minutes.
Etching each channel pattern on the glass substrate: after washing with ultrapure water, the photoresist-coated chrome plate with a clear micro-pattern was etched with an etching solution at 37 ℃ for 150 minutes. To avoid SPE particle leakage, a 20 μm high dam was fabricated into the microchannel. First, the ends of the pass-through SPE column were protected with 1 mm wide tape prior to the first etching step. After 150 minutes of etching, the protective tape was removed and etched under the same conditions for an additional 15 minutes to form a dam. And then ultrasonically damaging the photoresist on the surface by using acetone, putting the chromium layer into a chromium removing solution to remove the chromium layer, and finishing the glass mold with the channel. The resulting glass mold was then rinsed with ultrapure water and dried at 80 ℃.
(2) M1 and M2 module preparation: the pre-mixed PDMS prepolymer and curing agent (10: 1 mass ratio) were poured onto the clean glass substrate of step one, degassed in a vacuum chamber for 30 minutes, and then cured in an oven at 80 ℃ for 1.5 hours. The PDMS replica was carefully peeled off from the slide, corresponded to the glass mold prepared in (1), and punched using a flat-head syringe needle. The PDMS sheets were sealed after 60 seconds of oxygen plasma treatment on glass, and the chips integrated with the M1 and M2 modules were completed.
(3) Preparation of M3 modules in microfluidic chips: as shown in fig. 2, a PDMS film was prepared by spin-coating a layer of PDMS (prepolymer and curing agent in a weight ratio of 10:1) on a clean glass substrate and baking for 90 minutes at 80 ℃, the spin-coating procedure was: step 1(500r/min, 10s) and step 2(1500r/min, 30s), the film with a thickness of 40 μm was measured with a vernier caliper as the film layer. The top PDMS layer (with G channels) was first bonded to the membrane, and the membrane and G channels were carefully peeled apart together. The bottom glass layer (with L-channels) is bonded to the other side of the membrane to form a sandwich, requiring careful alignment of the G-channels with the L-channels.
Example 2
Optimizing the structure of the microfluidic chip:
in order to improve the overall analysis performance of the microfluidic chip, some important factors of the three modules are studied: flow rate of milk sample, spacing of the micro-column array in M1, filling amount of EMR-lipid particles in M2.
The flow rate is crucial to the extraction effect of the veterinary drug. To determine the optimal conditions for the newly developed on-line method, the effect of sample flow rate on extraction rate was investigated. Thus, the sample solution was passed through M1 at a flow rate of 10 to 80. mu.L/min. The theoretical concentration of the standard added milk is 50 ng/mL. The results show that the highest concentration was obtained at a flow rate of 10. mu.L/min; the sample flow rate was increased to 80 μ L/min and the extraction rate was reduced to only 16.8% (in the case of SM 2). This should be because a higher flow rate results in a shorter time for the sample and the extraction liquid to contact each other, and thus the extraction efficiency is reduced. The extraction rate of each drug was different. For example, the overall extraction efficiency of sulfonamides is only around 75%. Although some drugs have a higher extraction efficiency at 25 μ L/min, the final injection rate for the subsequent experiments was 10 μ L/min, taking all factors into account.
The arrangement pitch of the filter units is determined by the size of the impurity particles. In order to prevent the filter unit from being blocked by impurity particles, a filter with a micro-column array pitch reduced from 400 μm to 100 μm is adopted to obtain better filtering effect. The diameter of the micro-column array cylinder is 200 μm. Whether two columns are connected in series is also considered. The filtering unit structure is as follows: a: 400-300-200-100 μm, and the double columns are connected in series; b: 400-300-200 μm, and the double columns are connected in series; c: 400-300-200-100 μm, single column; d: 400-300-200 μm, single column. Comparing the weight of the co-extract in the extracts, the weight of the residue obtained in structure a was the smallest, indicating the best filtration. Clear extracts can be obtained with structure a without channel blockage.
A novel adsorbent, known as EMR-Lipid, selectively interacts with the unbranched hydrocarbon chains of the lipids, leaving the target analyte in solution for subsequent analysis. EMR-Lipid straight-through columns utilize this selective fat removal to further simplify the workflow and for multi-class, multi-residual pesticide and veterinary drug analysis in fat matrices. However, the application of the adsorbent to the conventional pretreatment method requires off-line nitrogen purging, and cannot achieve the purpose of automation. The loading of EMR lipid in the flow-through purification module varies from 0mg to 100 mg. The co-extraction residue removal rate (by weight) evaluated the sample purification efficiency by measuring the weight of the sample co-extract to remove matrix co-extract from the crude sample extract. An amount of 50mg EMR lipid had the highest removal rate of matrix co-extract, and had reached the same level as reported in other studies. Of course, if the amount of particles is increased, the removal of the matrix co-extract may be better. The amount of 50mg was chosen taking into account the size of the chip and the pressure of the fluid in the channels of the chip.
Example 3
Straight-through purification module (M2) effect on Matrix (ME) removal:
traditional HPLC-MS quantitative methods are based on chromatographic separations, time consuming and require specialized equipment and personnel. Our proposed method does not require chromatographic separation and therefore more effort must be made in on-line sample preparation. The response signal of MS is susceptible to influences from complex sample matrices, especially in ESI mode. However, most microfluidic chip-MS/MS methods published in the literature do not analyze or solve the ME problem, although the elimination of matrix effects is crucial to establishing a reliable method. Species of similar nature in the matrix may co-elute with and affect ionization of the target analyte, resulting in signal suppression or enhancement. Matrix matching calibration, isotope dilution, and sample dilution are proposed in the literature to estimate or compensate for matrix effects. An ME value of 100% indicates that no absolute matrix effect is observed. Values > 100% indicate increased ionization and values < 100% indicate suppressed ionization. ME is a key aspect to obtaining reliable results in MS analysis. ME can be classified as negligible (80% < ME < 120%), moderate (120% -150% or 50% -80%) and strong (ME > 150% or < 50%). Figure 3 shows the effect of integrated microfluidic chips on ME. ME is the average data for three different concentrations of matrix-matched standard solutions (5ng/mL, 20ng/mL, and 50 ng/mL). Without M2, the ME is essentially in the strong inhibition zone. After M2, the ME of 12 drugs shifted from strong to moderate inhibition, with 3 drugs negligible. The results show that EMR lipids used for decontamination significantly improved ME over those not decontaminated. ME may result from competition between analyte and co-eluting, undetected matrix components reacting with primary ions formed in the MS interface. EMR-lipid particles reduced the co-eluting matrix components, as evidenced by the matrix co-extract removal data.
The mechanism of interaction between the EMR-Lipid adsorbent and the undesirable lipids in the sample matrix is highly selective, and therefore EMR-Lipid purification does not typically result in retention of the target analyte. The present study excluded the effect of the extraction step on analyte recovery to evaluate analyte recovery before and after EMR-Lipid purification. The results show that EMR-Lipid does not result in significant analyte loss, with target recovery ranging from 80.0% to 100.2% after through-purification.
Although the ME is greatly increased by the pre-treatment, the peak shape is improved and the response of the target is increased. However, ME is still unavoidable. ME in MS analysis is present objectively and varies from matrix sample to matrix sample. Based on this, an Internal Standard (IS) was introduced for detection. IS can not only compensate ME during MS ionization, but also eliminate differences during sample preparation. The quinolone drugs use OFX-d3, the sulfonamides use SDM-d6, the macrolides and lincosamides use ROM-d7, and the antiviral drugs use ATD-d15 as IS. The ME of the matrix standard solution prior to cleaning was found to be relatively unstable when the ME was calculated by the IS method, and the error line data shows that the ME for all 9 targets exceeded 120%. The use of internal standards has proven to be conditional and must be used to purify relatively complete sample solutions, otherwise quantitative deviations can result. By comparing chromatograms before and after purification, the peak shape after EMR-lipid purification has no tailing, is more symmetrical and has higher intensity. Using EMR-lipid greatly improves the peak shape of both quantitative ionic and qualitative examples, as exemplified by the target IMQ. Fewer impurities were observed in the chromatogram and the intensity was doubled (as shown in fig. 4). Therefore, the peak area integrals are prone to bias without passing through the purge due to more substrate interference prior to purge. All indices for the purified matrix standard solution ME calculated by IS method were within the negligible ME range shown in figure 1, indicating that the difference between the pure solution standard and the matrix-matched standard was essentially negligible. Therefore, IS compensates significantly for ME. Quantitative calculation in subsequent experiments is carried out by combining a pure solution standard curve with an internal standard method instead of a matrix matching standard.
Example 4
Online osmotic solvent evaporation:
m3 in the integrated microfluidic chip for sample pervaporation has a multilayer structure with L channels, membranes and G channels (fig. 3). The perforation position of the L channel is staggered with the G channel so as to ensure that the upper channel and the lower channel are not interfered with each other. PDMS films are very thin and easily deformed. Therefore, in the production process, the solidified film spin-coated on the glass substance is firstly bonded with the gas layer (G-channel) to form a sandwich structure, and then the film and the G-channel are peeled together to be bonded with the L-channel, so that the production success rate can be greatly improved. The bonded chip is enclosed and connected to N2. The flow directions of the liquid and gas are kept counter-current rather than co-current, and the evaporation efficiency obtained in the counter-current case is higher than that obtained in the co-current case. The reason is that in chemical engineering, countercurrent flow provides a higher average driving force for heat and mass transfer than co-current flow.
Since the saturation pressure of each component is temperature dependent, the operating temperature is expected to affect pervaporation efficiency. Permeability is inversely proportional to the thickness of the membrane. To maximize permeability, minimization of the membrane thickness and increasing the membrane surface area and residence time will positively impact the pervaporation process. With a fixed flow rate, the operating temperature and the membrane thickness are optimized. The evaporation rate was calculated by comparing the volume change of the inlet and outlet samples. FIG. 5 shows that the evaporation rate of films of different thicknesses increases significantly with increasing temperature. When the temperature reaches 60 ℃, the films of 40 μm and 70 μm can completely evaporate all the organic solution, and the evaporation efficiency is highest at 70 ℃. However, if the temperature is too high, the drug may be lost by decomposition. Comparing the recovery rates of drugs at different temperatures, the losses of sulfonamides, macrolides and some antiviral drugs increased when the temperature reached 70 ℃, especially the average loss rate of sulfonamides reached 21% (see fig. 6). The data in fig. 5 also demonstrate that the thinner the film, the faster the evaporation rate. In our experiments, PDMS films below 30 μm appear very fragile, stick easily to glass slides and are difficult to handle. The temperature and the thickness of the film were thus set to 60 ℃ and 40 μm.
By the design of the evaporation module in the integrated microfluidic chip and the combination of the previously designed mixed extraction, filtration and pass-through module, the response intensity of the target object can be obviously improved. The evaporation performance was exhibited by comparing the response intensity of the target before and after the chip M3. Two representative signal intensity profiles are given to show the effect of evaporation. FIG. 7 shows the detection signal before and after the labeled milk passes through the chip evaporation module. ACV gave no signal before evaporation at a spiked concentration of 15ng/mL, whereas the detection signal was obtained after passage through the chip. The peak intensity of SM2 at a spiked concentration of 15ng/mL increased 6-fold after passing through the evaporation chip.
Example 5
The combination of the full-automatic sample pretreatment micro-fluidic chip and the mass spectrum:
the reliability of quantitative analysis using MS/MS may be adversely affected by lack of sensitivity due to ME-induced ion suppression, and most methods tend to use matrix-matched standard curves for quantitation to avoid adverse effects of the matrix. The integrated chip designed in the research purifies the milk sample by precipitating protein and removing fat, eliminates matrix interference, and improves the detection sensitivity by online evaporation. The calculation using standard solutions instead of matrix solutions can greatly simplify the experimental procedure due to the calibration effect of the Internal Standard (IS). The standard curve is plotted from a series of standard solution concentrations (0.5-50ng/mL, internal standard 10ng/mL) and peak area after evaporation of the target using internal standard method. The standard working curve shows that the quantitative performance of the IS method IS relatively good (R2 > 0.99).
In order to realize the on-line quantitative analysis of the multi-residue veterinary drug, an Internal Standard (IS) method IS used on the full-automatic microfluidic chip-ESI-MS platform. Precision and accuracy are of great importance to evaluation method development and validation. During the whole analysis process, three different concentrations of standard and internal standard were added to the milk, the concentration of the internal standard being consistent with that in the standard curve. The spiked milk and extract were pumped simultaneously into the developed automated microfluidic chip. Through an on-line extraction, filtration (M1) and through-type purification module (M2), and finally into an evaporation module (M3). The solution was collected by a collection ring and introduced directly to the MS detector through a switching valve. The automatic operation system does not need any off-line manual operation, the whole analysis and detection time of one sample is less than 30 minutes, and the time is shortened by about 10 times compared with the traditional LC-MS/MS method. Furthermore, the consumption of sample and reagents is about 20 times less than in the conventional LC-MS/MS method.
The sample solution was analyzed directly by MS without chromatographic separation. To reduce the matrix effect, the volume of the milk sample was only 100 μ L. Due to the efficient purification of EMR-lipid particles and the use of an evaporation module (M3), the limit of detection (LOD) of the spiked milk samples based on signal-to-noise ratio (S/N ═ 3) was in the range of 0.23-4.13 ng/mL. . The limit of quantitation (LOQ) for methods based on signal-to-noise ratio (S/N ═ 10) is in the range of 0.76-13.7 ng/mL. According to the national standard (GB 31650-. Table 1 shows the recovery and RSD of different targets on the developed microfluidic chip-MS platform.
Table 123 linear equations for targets, R2, LOD, LOQ, MRL and recovery (n ═ 3)
The absence of a MRL indicates that the maximum allowable residual limit b ND of these compounds has not been established in milk indicates that these compounds are not allowed to be detected in animal products.
As shown in table 1, the average recovery of the analytes was 71.7% to 118.0% and the RSD was 1.9% to 15.2% at spiked concentrations of 5, 15 and 50ng/mL of milk. The recovery and RSD were essentially satisfactory. The developed microfluidic-MS platform has the advantages of rapidness, simplicity and automation, and is a rapid screening method for multi-residue analysis. The principle of the MRM module in tandem quadrupole mass spectrometry is that a first quadrupole is used to mass select precursor ions, fragmented in a collision cell, and then a second quadrupole is used to mass select specific product ions, which has gained wide acceptance due to its high selectivity and sensitivity. The MRM ratio in the method completely meets the requirement, and the feasibility of the chip-MS method is further explained. The developed full-automatic microfluidic chip-MS platform can be effectively and simultaneously used for qualitative and quantitative detection of multiple targets. The core of the system is a micro-fluidic chip containing EMR lipid particles for purification and a PDMS membrane for evaporation. The organic solvent in the sample was removed by in-line evaporation to concentrate the target. The three serial modules in the microfluidic chip were designed to remove as much as possible of the substrate interferences in milk and allow on-line detection by MS without chromatographic separation. And the system can be extended to the detection of various food samples.
The integrated microfluidic system has the advantages of high sensitivity, high detection speed, accurate detection result, simple operation, less sample dosage, less reagent dosage and the like, and has remarkable potential in the aspect of multi-class and multi-residue detection. In addition, microfluidic systems will be a powerful tool as the best quantitative screening method for food safety. In general, the designed system can ensure the quality of milk, thereby playing a great application potential in the future.
Claims (10)
1. A micro-fluidic chip for online pretreatment of samples is characterized by comprising an automatic extraction and filtration module, a pass-through purification module and an online pervaporation module which are sequentially communicated; the automatic extraction and filtration module is used for removing particle impurities in the sample, and the through type purification module is used for removing fat and phospholipid in the sample; the online pervaporation module is used for removing the solvent generated by the automatic extraction and filtration module.
2. The microfluidic chip for online pretreatment of samples according to claim 1, wherein the automatic extraction and filtration module is a micro-column array with a spacing of 100-400 μm.
3. The microfluidic chip for online sample pretreatment according to claim 1, wherein the purification module has a three-channel parallel structure, and the channels are filled with enhanced matrix to remove lipid particles.
4. The microfluidic chip for online pretreatment of samples according to claim 3, wherein each channel has a length of 3-10cm and a width of 4-10mm, and the filling amount of the enhanced matrix removal lipid particles of each channel is 10-100 mg.
5. The microfluidic chip for online pretreatment of samples according to claim 1, wherein the online pervaporation module has a multi-layer structure comprising a liquid layer, a membrane layer and a gas flow layer, wherein the liquid layer and the gas flow layer have the same channel pattern and are both serpentine channel patterns.
6. The microfluidic chip for online sample pretreatment according to claim 5, wherein the flow directions of the liquid in the liquid layer and the gas in the gas layer are opposite.
7. The microfluidic chip for online pretreatment of samples according to claim 5, wherein the online pervaporation module is disposed on the heating plate, and the inlet of the gas flow layer is connected with an inert gas.
8. A method for preparing a microfluidic chip according to any one of claims 1 to 7, comprising the steps of:
(1) transferring the channel patterns of the automatic extraction and filtration module, the pass-through purification module and the online pervaporation module on a glue-homogenizing chromium plate for etching, and then removing a chromium layer to obtain a glass substrate with channel patterns with a certain depth;
(2) pouring the PDMS prepolymer into another smooth glass substrate, curing PDMS, stripping the PDMS replica from the glass mold, perforating and sealing the position corresponding to the glass substrate with the channel to obtain a chip integrating the automatic extraction and filtration module and the straight-through purification module;
(3) and (3) spin-coating PDMS on a clean glass substrate to obtain a PDMS film with a certain thickness, bonding the PDMS film with a PDMS gas layer with a serpentine channel, peeling the PDMS film and the film layer from the glass substrate, and bonding the other surface of the film layer with the serpentine channel of the glass substrate to obtain the microfluidic chip.
9. A method of using a microfluidic chip according to any of claims 1 to 7, wherein the amount of drug remaining in the sample is determined by combining the microfluidic chip with a biochip and/or mass spectrometry.
10. The use method of the microfluidic chip according to claim 9, wherein when the sample is milk and the component to be measured is a veterinary drug, the online pervaporation module is set to operate at 60-70 ℃ and the thickness of the membrane layer is 40-70 μm.
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