CN108088822B - Chip type light derivatization device for aflatoxin and sulfanilamide drugs - Google Patents
Chip type light derivatization device for aflatoxin and sulfanilamide drugs Download PDFInfo
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Abstract
The invention relates to a chip type light derivatization device for aflatoxin and sulfonamides, which adopts an ultraviolet Light Emitting Diode (LED) with the central wavelength of 280-380nm as an excitation light source, adopts highly transparent quartz, ultraviolet-transmitting glass, PDMS or perfluoroethylene propylene copolymer (FEP) as a chip substrate, and manufactures a rectangular or trapezoidal groove with the width of 10-500 microns, the depth of 10-260 microns and the length of 5-500mm on the chip substrate as a derivatization reaction tank, wherein the groove is arranged in a linear or comb or sine wave shape, the volume of the derivatization tank is 5nL-75 muL and is only 1/13 to one hundred thousand of the volume of the traditional derivatization device. The chip type optical derivatization device disclosed by the invention can improve the fluorescence intensity of aflatoxin B1 by 6.5 times, and is suitable for post-column optical derivatization and micro-flow injection analysis/small-size flow injection analysis of chip capillary electrophoresis, chip micro-fluidic analysis, capillary liquid chromatography, micro-column liquid chromatography, ultra-high pressure liquid chromatography and conventional liquid chromatography.
Description
Technical Field
The invention relates to the field of optical detection of microfluidic chemical analysis, capillary high performance liquid chromatography (mu-HPLC) and microfluidic injection analysis (mu-FIA), in particular to a light derivatization device for aflatoxin and sulfonamides.
Background
The light derivatization device is widely used for quantitative detection of aflatoxin and sulfanilamide drugs by a High Performance Liquid Chromatography (HPLC) -fluorescence detection method, such as national standards GB/T18979-. With the development of times and the progress of science and technology, the limit value of national standards and European Union detection standards on aflatoxin in various substances is gradually reduced, the difficulty of analysis and detection is continuously improved, the lower limit of detection is lower than the level of a conventional fluorescence detector, and the detection sensitivity must be improved by means of derivatization or molecular marking technology. In recent decades, green chemical concepts and technologies have been developed and deeply concentrated, and the aim is to fundamentally solve the problems of environmental pollution and health and safety of laboratory personnel. Accordingly, in the field of analytical chemistry, it is the current direction of development to develop analytical equipment and techniques that reduce reagent usage by 90% or even 99% over existing equipment. Microfluidic analytical techniques have also been developed.
However, micro-flow analysis instruments require that the cell volume of the detector is also small, for example, chip micro-fluidic analysis and micro-fluidic injection analysis require that the detection cell volume is 2.5 nL-70 nL, capillary high performance liquid chromatography requires that the detection cell volume is 20 nL-500 nL, and micro-column liquid chromatography requires that the detection cell volume is 0.5 muL-2 muL. From the aspect of analysis and detection, the smaller the cell volume of the derivatization device is, the better the cell volume is; however, the smaller the cell volume, the lower the amount of light radiation and the shorter the derivatization reaction time, which leads to a decrease in derivatization efficiency. At present, the high derivatization efficiency is exchanged by sacrificing the separation efficiency of the chromatographic column and allowing the chromatographic peak to be broadened in the conventional analysis, for example, a fluorescence detector with a cell volume of only 12 μ L is matched with a 1mL light derivatization device with a derivatization cell volume which is 83 times that of the detector cell! However, with ultra-high pressure liquid chromatography (UPLC), the dead volume outside the column is very demanding and so large cell volumes of derivatizers and detectors cannot be used, the sum of which cannot exceed 10 μ L. At present, no light derivatization device with a cell volume of several microliters and high derivatization efficiency is reported. For microfluidic analysis and capillary liquid chromatography, the cell volume of the post-column derivatization device can only be less than or equal to the detection cell volume, otherwise the separation efficiency is affected.
However, the aflatoxin light derivatization device used up to now, whether it is used for scientific research (home-made photochemical derivatization reactor of fang hai east, wang ling feng, zhao ji-zhao-shi "measuring aflatoxin B1 in soybean oil by high performance liquid chromatography, physicochemical examination-chemical division book, 2013, 49, 999-. For the post-column optical derivatization of chip capillary electrophoresis, chip microfluidic analysis, micro-flow injection analysis, capillary liquid chromatography, micro-column liquid chromatography and ultrahigh pressure liquid chromatography, no report of an optical derivatization device with the volume from nanoliter to micro-upgrading pool is reported at present.
Disclosure of Invention
Aiming at the problems, in order to improve the defects of the prior art and based on further and intensive research on a photochemical derivatization reaction mechanism, the invention provides that an ultraviolet LED light source with the central wavelength of 280-380nm is adopted to replace a traditional low-pressure mercury lamp as an excitation light source; the method comprises the following steps of (1) replacing a traditional derivative reaction tube with a net-shaped woven structure by a linear or square wave or sine wave-shaped arranged light derivative reaction channel on a chip, wherein the reaction channel is a rectangular or trapezoidal channel with the width of 10-500 mu m, the depth of 10-260 mu m and the length of 5-500mm, and the volume of a derivative pool is only 5nL-75 mu L; experiments prove that the fluorescence intensity of the aflatoxin G1 can be improved by 7 times and the fluorescence intensity of the aflatoxin B1 can be improved by 6 times by adopting the 7.5 mu L cell volume light derivatization device, and the efficiency is similar to that of the traditional 1000 mu L cell volume light derivatization device.
The technical scheme of the invention is as follows:
the utility model provides an aflatoxin and sulfanilamide medicine's chip formula light derivatization ware comprises ultraviolet LED light source, chip derivatization reaction passageway, chip base plate, reflector panel, LED base plate and fin, its characterized in that:
one or more than two ultraviolet LED light sources which are arranged along a straight line are positioned above the chip derivatization reaction channel and are used as excitation light sources for the light derivatization reaction; one or more than two ultraviolet LED light sources are welded on the lower surface of the LED substrate; the ultraviolet LED light source is positioned above the derivative reaction tube, and the lower surface of the LED substrate is parallel to the plane of the rectangular area of the chip derivative reaction channel; one or more than two ultraviolet LED light sources which are arranged along a straight line are positioned in the straight line and are parallel to the chip derivatization reaction channel in the rectangular area, and the projection of the straight line of the ultraviolet LED light sources on the plane of the rectangular area passes through the center of the rectangular area of the chip derivatization reaction channel; a certain distance is arranged between the ultraviolet LED light source and the chip derivation reaction channel;
the chip derivation reaction channel is arranged on the chip substrate, is a rectangular or trapezoidal groove and is arranged in a linear, square-wave or sine-wave shape; the chip substrate is made of highly transparent quartz, ultraviolet-transmitting glass, PDMS or FEP (perfluoroethylene propylene copolymer);
the reflecting plate is of a structure with a plane upper surface, is positioned below the chip derivatization reaction channel, is parallel to the plane of the rectangular area of the chip derivatization reaction channel, and is arranged at a certain distance from the chip derivatization reaction channel; the radiating fins are tightly attached to the upper surface of the LED substrate and used for radiating the ultraviolet LED light source.
The central wavelength of the ultraviolet LED light source is between 280 and 380nm, and the divergence angle is 60-140 degrees; the ultraviolet LED light source and the chip derivatization reaction channel are arranged at a certain distance, namely a gap is reserved between the ultraviolet LED light source and the chip derivatization reaction channel, so that the ultraviolet LED light source uniformly irradiates the chip derivatization reaction channel; the distance between the ultraviolet LED light source and the chip derivation reaction channel is 0.1-7 mm.
The width of the chip derivative reaction channel is 10-500 micrometers, the depth is 10-260 micrometers, the length is 5-500 millimeters, and the internal volume is 5 nanoliter-75 microliters; the reflecting plate and the chip derivative reaction channel are arranged at a certain distance, namely a gap is reserved between the reflecting plate and the chip derivative reaction channel to improve the reflecting efficiency; the distance between the chip derivation reaction channel and the reflecting plate is 0.1-7 mm.
The material of reflector panel is for polishing stainless steel board or polishing aluminum plate, and the reflector panel has the heat dissipation function simultaneously.
The LED substrate and the radiating fins are made of metal materials with good heat conduction, such as aluminum or copper, and the LED substrate and the radiating fins are tightly attached to facilitate heat dissipation.
One or more than two ultraviolet LED light sources are welded on the LED substrate and have the same height after being welded, namely the lower ends of the ultraviolet LED light sources are positioned on the same horizontal plane.
Compared with the prior art, the photochemical derivative has the following advantages:
1. the traditional light derivatization device adopts a woven derivatization reaction tube, the volume of the cell is 100-1000 mu L, and the traditional light derivatization device cannot be applied to post-column derivatization in microfluidic analysis and capillary liquid chromatography analysis; the invention adopts the thin-inner-diameter derivatization reaction tube which is arranged in a linear or comb or sine wave shape, the volume of the reaction tank is only 5nL-75 muL, the volume of the reaction tank is matched with a microfluidic analysis system, the light derivatization efficiency is greatly improved, and the cost is reduced;
2. the traditional light derivatization device adopts a 254nm mercury lamp ultraviolet light source, while the invention adopts a 280-380nm central wavelength LED ultraviolet light source, and still obtains high derivatization efficiency. For the LED with the central wavelength of 355 to 380nm, the service life is more than 20000 hours, the total power consumption of the ultraviolet LED light source is only 1-3W, and the ultraviolet LED light source is far superior to the traditional low-pressure mercury lamp in terms of service life, stability, power consumption, volume and even cost indexes.
3. The volume of a derivatization pool of the chip type optical derivatization device is only 5nL-75 muL, so that post-column derivatization of chip-level microfluidic analysis and capillary liquid chromatography analysis is realized. The optical derivatization device is suitable for post-column optical derivatization and micro flow injection analysis/small flow injection analysis of chip capillary electrophoresis, chip microfluidic analysis, capillary liquid chromatography, micro-column liquid chromatography, ultrahigh pressure liquid chromatography and conventional liquid chromatography, and improves the detection sensitivity.
4. The chip photochemical derivatization device has the advantages of small cell volume, high photo-derivatization efficiency, low cost, small volume, effective improvement of the detection sensitivity of detecting aflatoxin and sulfonamides, and strong practicability.
Drawings
FIGS. 1(a) and 1(b) are two views of a chip-based photo-derivatizer, wherein: (a) is a vertical cross-sectional view, and (b) is a top view of the chip-derived reaction channel. In the figure: 1-ultraviolet LED light source, 2-chip derivative reaction channel, 3-chip substrate, 4-reflector, 5-LED substrate, and 6-heat sink.
FIG. 2 is a detection spectrum of example 1.
Detailed Description
The utility model provides an aflatoxin and sulfanilamide medicine's chip formula light derivatization ware, comprises ultraviolet LED light source 1, chip derivatization reaction passageway 2, chip base plate 3, reflector panel 4, LED base plate 5 and fin 6, its characterized in that:
one or more than two ultraviolet LED light sources 1 which are arranged along a straight line are positioned above the chip derivatization reaction channel 2 and are used as excitation light sources for the light derivatization reaction; one or more than two ultraviolet LED light sources 1 are welded on the lower surface of the LED substrate 5; the ultraviolet LED light source 1 is positioned above the derivative reaction tube 2, and the lower surface of the LED substrate 5 is parallel to the plane of the rectangular area of the chip derivative reaction channel 2; one or more than two ultraviolet LED light sources 1 which are arranged along a straight line are positioned in the straight line and are parallel to the chip derivation reaction channel 2 in the rectangular area, and the projection of the straight line of the ultraviolet LED light sources 1 on the plane of the rectangular area passes through the center of the rectangular area of the chip derivation reaction channel 2; a certain distance is arranged between the ultraviolet LED light source 1 and the chip derivation reaction channel 2;
the chip derivation reaction channel 2 is arranged on the chip substrate 3, is a rectangular or trapezoidal groove path and is arranged in a linear, square wave or sine wave shape; the material of the chip substrate 3 is highly transparent quartz, ultraviolet-transmitting glass, PDMS or FEP (perfluoroethylene propylene copolymer);
the reflecting plate 4 is a structure with a plane upper surface, the reflecting plate 4 is positioned below the chip derivatization reaction channel 2, the upper surface of the reflecting plate 4 is parallel to the plane of the rectangular area of the chip derivatization reaction channel 2, and a certain distance is arranged between the reflecting plate 4 and the chip derivatization reaction channel 2; the radiating fins 6 are tightly attached to the upper surface of the LED substrate 5 and used for radiating the ultraviolet LED light source.
The central wavelength of the ultraviolet LED light source 1 is between 280-380nm, and the divergence angle is 60-140 degrees; a certain distance is reserved between the ultraviolet LED light source 1 and the chip derivatization reaction channel 2, namely, a gap is reserved between the ultraviolet LED light source 1 and the chip derivatization reaction channel 2, so that the ultraviolet LED light source 1 uniformly irradiates the chip derivatization reaction channel 2; the distance between the ultraviolet LED light source 1 and the chip derivation reaction channel 2 is 0.1-7 mm.
The width of the chip derivative reaction channel 2 is 10-500 microns, the depth is 10-260 microns, the length is 5-500mm, and the internal volume is 5 nanoliter-75 microliters; a certain distance is arranged between the reflector 4 and the chip derivative reaction channel 2, namely a gap is reserved between the reflector and the chip derivative reaction channel for improving the reflection efficiency; the distance between the chip derivation reaction channel 2 and the reflecting plate 4 is 0.1-7 mm.
The material of the reflector 4 is a polished stainless steel plate or a polished aluminum plate, and the reflector has a heat dissipation function.
The LED substrate 5 and the heat sink 6 are made of metal material with good heat conduction, such as aluminum or copper, and the LED substrate 5 and the heat sink 6 are tightly attached to each other, so that heat dissipation is facilitated.
One or more than two ultraviolet LED light sources 1 are welded on the LED substrate 5 and have the same height after being welded, namely the lower ends of the ultraviolet LED light sources 1 are positioned on the same horizontal plane.
Example 1
An ultra-high performance liquid chromatography (UPLC) post-column light derivatization device is characterized in that an excitation light source is 2 high-power ultraviolet LEDs with central wavelength of 365nm, the divergence angle is 120 degrees, and the rated current is 500 mA; the distance between the ultraviolet LED light source and the chip derivative reaction channel is 3 mm; the chip substrate is made of high-transparency quartz glass, and is provided with chip derivation reaction channels which are arranged in a comb shape, the channels are rectangular groove paths with the width of 150 mu m, the depth of 200 mu m and the length of 250mm, and the volume of the derivation pool is 7.5 mu L; the distance between the chip derivative reaction channel and the reflector is 3 mm; the reflecting plate is a stainless steel polishing plate; the LED substrate and the radiating fins are made of metal aluminum. The performance of the chip-based optical derivatiser was evaluated by coupling it to a Waters e2475 FLD-type fluorescence detector (2. mu.L cell volume). The liquid chromatography conditions were: a chromatographic column: c18 column, 100mm × 2.1mm × 1.7 μm; mobile phase a methanol: mobile phase B pure water 45: 55; flow rate of mobile phase: 0.3 mL/min; sample introduction: 10 μ L of 0.6ppb aflatoxin.
The experimental results are as follows:
FIG. 2 is a detection spectrum: (1) directly injecting sample, carrying out chromatographic separation without derivation, detecting by using Waters e2475FLD, wherein the lower limit of detection on aflatoxin B1 is 0.14 ppb; (2) the chip type optical derivatization device is connected between the outlet of the chromatographic column and the inlet of the detector in series, the detection sensitivity of the chip type optical derivatization device to the aflatoxin B1 is improved by 6.5 times, and the detection limit is 0.02 ppb.
Example 2
The chip-based optical derivatization device described in example 1 was used in combination with a Waters e2475FLD model fluorescence detector to detect 4 sulfonamides. Excitation wavelength 230nm, emission wavelength 400 nm.
The experimental results are as follows: injecting 5ppb Sulfadiazine (SDZ), and detecting no Waters e2475FLD when a derivatization device is not connected; after the chip type optical derivatization device is connected, 5ppb Sulfadiazine (SDZ) can be detected, and the detection signal-to-noise ratio is 15.
It will be apparent to those skilled in the art that modifications may be made in the details of this embodiment and its scope of application without departing from the spirit of the invention. The present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the general principles and novel features disclosed herein. Any changes that are made without inventive step in the light of the present inventive concept are within the scope of the present invention.
Claims (7)
1. The utility model provides an aflatoxin and sulfanilamide medicine's chip formula light derivatization ware comprises ultraviolet LED light source (1), chip derivatization reaction passageway (2), chip base plate (3), reflector panel (4), LED base plate (5) and fin (6), its characterized in that:
one or more than two ultraviolet LED light sources (1) which are arranged along a straight line are positioned above the chip derivatization reaction channel (2) and are used as excitation light sources for the light derivatization reaction; one or more than two ultraviolet LED light sources (1) are welded on the lower surface of the LED substrate (5); the ultraviolet LED light source (1) is positioned above the derivatization reaction tube (2), and the lower surface of the LED substrate (5) is parallel to the plane of the rectangular area of the chip derivatization reaction channel (2); one or more than two ultraviolet LED light sources (1) arranged along a straight line are positioned in the straight line and are parallel to the chip derivation reaction channel (2) in the rectangular area, and the projection of the straight line of the ultraviolet LED light sources (1) on the plane of the rectangular area passes through the center of the rectangular area of the chip derivation reaction channel (2); a certain distance is arranged between the ultraviolet LED light source (1) and the chip derivation reaction channel (2);
the chip derivation reaction channel (2) is arranged on the chip substrate (3), is a rectangular or trapezoidal groove, and is arranged in a linear, square wave or sine wave shape; the chip substrate (3) is made of highly transparent quartz, ultraviolet-transmitting glass, PDMS or FEP;
the reflecting plate (4) is of a structure with a plane upper surface, the reflecting plate (4) is positioned below the chip derivatization reaction channel (2), the upper surface of the reflecting plate (4) is parallel to the plane of the rectangular area of the chip derivatization reaction channel (2), and a certain distance is arranged between the reflecting plate (4) and the chip derivatization reaction channel (2); the radiating fins (6) are tightly attached to the upper surface of the LED substrate (5) and used for radiating the ultraviolet LED light source;
the width of the chip derivation reaction channel (2) is 10-500 micrometers, the depth is 10-260 micrometers, the length is 5-500 millimeters, and the internal volume is 5 nanoliter-75 microliters; the distance between the chip derivation reaction channel (2) and the reflecting plate (4) is 0.1-3 mm; the distance between the ultraviolet LED light source (1) and the chip derivation reaction channel (2) is 0.1-3 mm.
2. The chip-based photodeivator of claim 1, wherein: the central wavelength of the ultraviolet LED light source (1) is between 280 and 380nm, and the divergence angle is 60-140 degrees; the ultraviolet LED light source (1) and the chip derivatization reaction channel (2) are arranged at a certain distance, namely a gap is reserved between the ultraviolet LED light source (1) and the chip derivatization reaction channel (2), so that the ultraviolet LED light source (1) uniformly irradiates the chip derivatization reaction channel (2).
3. The chip-based photodeivator of claim 1, wherein: the reflecting plate (4) and the chip derivative reaction channel (2) are arranged at a certain distance, namely a gap is reserved between the reflecting plate and the chip derivative reaction channel for improving the reflecting efficiency.
4. The chip-based photodeivator of claim 1, wherein: the material of reflector panel (4) is for polishing stainless steel board or polishing aluminum plate, and the reflector panel has the heat dissipation function simultaneously.
5. The chip-based photodeivator of claim 1, wherein: the LED substrate (5) and the radiating fins (6) are made of metal materials with good heat conduction, and the LED substrate (5) and the radiating fins (6) are tightly attached to facilitate heat dissipation.
6. The chip-based photodeivator of claim 5, wherein: the metal material is aluminum or copper.
7. The chip-based photodeivator of claim 1, wherein: one or more than two ultraviolet LED light sources (1) are welded on the LED substrate (5) and have the same height after being welded, namely the lower ends of the ultraviolet LED light sources (1) are positioned on the same horizontal plane.
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| CN108931511B (en) * | 2018-06-04 | 2023-11-14 | 常州市武进区半导体照明应用技术研究院 | Ultraviolet LED fluorescence detection device and method |
| CN109406705B (en) * | 2018-11-29 | 2024-04-09 | 南京大学 | Liquid chromatography combined organic carbon detector and application method thereof |
| CN109358128B (en) * | 2018-12-03 | 2024-04-09 | 南京大学 | Organic nitrogen-organic carbon serial on-line detection method and device |
| CN109991346A (en) * | 2019-04-18 | 2019-07-09 | 南京大学 | A kind of micro-fluidic Ultraviolet Oxidation device for organic nitrogen analysis |
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