KR101466301B1 - Microfluidic chips for continuous monitoring of chromium in water having chemiluminesence measurement apparatus having thereof - Google Patents

Abstract

According to an aspect of the present invention, there is provided a microfluidic chip comprising: a reagent mixing channel for mixing a sample with a reagent and a reagent mixing channel for mixing the reagent and a reagent mixture channel; a reductant channel and a reagent mixing channel are connected in parallel at one end, And a second substrate coupled to the first substrate, the first substrate having a detection channel formed thereon, and a detection unit facing the detection channel and configured to transmit light.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a microfluidic chip for continuously monitoring chromium in water, and a chromium detection device including the microfluidic chip. [0002]

The present invention relates to a microfluidic chip for continuously monitoring the concentration of chromium present in water using a chemiluminescence method and a chromium detection device including the microfluidic chip.

Chromium is mainly used in alloys, pigments, leather, textile industry, catalysts, wood preservatives, etc. Because of its chemical stability, it is widely used as plating material to prevent metal corrosion. As the above-mentioned industrial activities increase, surface water and groundwater are contaminated due to the release of chromium.

Chromium ions in natural state exist in various oxidation states, but only the most stable chromium [III] and chromium [IV] exist in water. Chromium [III] is deficient in the essential nutrients involved in glucose, cholesterol, and fat metabolism, which reduces body weight and impairs the ability to remove glucose from the blood. And if exposed to skin for a long time, it may cause allergy or cancer. However, it is known that the toxicity is not high due to low solubility in water and permeability to biomembrane. On the other hand, chromium [IV] is known to have high solubility and mobility in water, high oxidative power and high permeability to biological membranes, which adversely affects various organs such as kidney, liver and lungs and causes inflammation of the skin or mucosa .

Because of the different properties and toxicity of chromium [III] and chromium [IV] as described above, it is very important to analyze their respective concentrations accurately. Particularly, chromium [III] and chromium [IV] are easily changed by oxidation-reduction reaction depending on the environment, so continuous monitoring of water quality is needed. In the case of Korea, the concentration of chromium [IV] in the clean area is regulated to 0.5 ppm and the concentration of chromium [IV] in the rest area is 0.1 ppm, And other areas are limited to less than 0.5 ppm. The US Environmental Protection Agency (EPA) defines chromium (IV) as a carcinogen and limits the total chromium concentration in drinking water to less than 0.1 ppm.

As a conventional technique for analyzing chromium in water, chromium analysis using a chemiluminescence reaction is carried out using luminol (5-amino-2-ene) oxidized by hydrogen peroxide under a basic condition of chromium [III] 3-dihydro-1,4-phthalazinedione). At this time, since the intensity of emitted light is proportional to the concentration of chromium [III], chromium [III] can be quantitatively analyzed by measuring the intensity of light.

[Formula 1]

In the case of chromium [IV], chromium [IV] is reduced to chromium [III] using a reducing agent under acidic conditions, since it does not directly participate in the chemiluminescence reaction of luminol. The reaction in which chromium [IV] is reduced to chromium [III] is shown in [Equation 2].

[Formula 2]

The method of analyzing chromium [III] and chromium [IV] by chemiluminescence method is as follows. First, the sample is reacted with a reducing agent to reduce chromium [IV] to chromium [III] in the sample, and the concentration of total chromium is measured. After measuring the concentration of chromium [III] without using a reducing agent, the concentration of chromium [IV] can be determined by calculating the difference in total chromium and chromium [III] concentration. The prior art has developed a microfluidic chip for chromium analysis in water by applying the above method, but has the following problems.

Firstly, the oxidation reaction of hydrogen peroxide with hydrogen peroxide in basic condition is very fast. Therefore, to efficiently measure the maximum chemiluminescence, it is necessary to minimize the length of the chemiluminescent reagent mixing channel in which luminol and hydrogen peroxide are mixed. In order to accurately analyze the concentration of chromium [IV], a sufficient reaction time is required as 100% reduction of chromium [IV] into chromium [III] in the sample is required. Therefore, the length of the reductive channel should be designed to be longer than the length of the chemiluminescent reagent mixing channel. However, in the prior art, the concentration of chromium [III] and chromium [IV] can not be accurately detected by making the length of the chemiluminescent reagent mixing channel equal to the length of the reducing channel for reduction of chromium [IV] to chromium [III] , The detection sensitivity decreases.

Secondly, the chemiluminescence reaction using luminol occurs not only with chromium [III] but also with other metal ions such as iron [II], cobalt [II], copper [II], nickel [II] The prior art does not take into account the effect on such interfering substances and thus can not selectively analyze the concentration of chromium in the sample.

Thirdly, mixing of chromium [III] with metal ions is caused by the pH change of the sample during mixing with the distilled water injected in the analysis of chromium [III] in the sample where other metal ions are present, and chromium [III] Precipitated co-precipitation may occur. When a coprecipitation phenomenon occurs, the concentration of chromium [III] in the sample injected into the microfluidic chip is reduced to cause an analysis error, resulting in clogging of the microfluidic chip due to the precipitation.

Fourth, two calibration curves (calibration curve of total chromium and chromium [III]) are needed to analyze chromium [III] and chromium [IV].

Fifth, as the microfluidic chip is made of a transparent glass substrate and ambient light enters the detector, the detection sensitivity is lowered and the operation of the apparatus becomes difficult.

The present invention detects chromium [III] and chromium [IV] with high efficiency and high sensitivity without continuous separation on a continuous flow to continuously monitor chromium in water by chemiluminescence method and selectively detects chromium And a chromium detection device having the same.

According to an aspect of the present invention, there is provided a microfluidic chip comprising: a reagent mixing channel for mixing a sample with a reagent and a reagent mixing channel for mixing the reagent and a reagent mixture channel; a reductant channel and a reagent mixing channel are connected in parallel at one end, And a second substrate coupled to the first substrate, the first substrate having a detection channel formed thereon, and a detection unit facing the detection channel and configured to transmit light.

The first and second substrates may be formed of a material having a color capable of absorbing light or may be colored with a color capable of absorbing light. have.

A sample inlet for injecting a sample and a reducing agent inlet for injecting a reducing agent are formed on the first substrate, the sample inlet is connected to a reducing channel via a sample channel, and the reducing agent inlet is connected to a reducing channel And a first reagent injection port for injecting the first reagent and a second reagent injection port for injecting the second reagent are formed on the first substrate, and the first reagent injection port is connected to the reagent through the first reagent channel And the second reagent inlet may be connected to the reagent mixing channel through the second reagent channel.

The detection channels may be overlapped and one end and the other end may be alternately connected to each other, and may be formed so as to decrease from the center to both ends. The reducing channel and the reagent mixing channel may be superimposed and one end and the other end Can be alternately connected.

A reagent mixing channel for mixing reagent and a reducing channel for mixing a sample and a reagent; and a reagent mixing channel for connecting the reducing channel and the reagent mixing channel in parallel at one end and a discharge port A microfluidic chip including a first substrate having a detection channel connected thereto and a second substrate coupled to the first substrate, the microfluidic chip having a detection unit formed to allow light to pass therethrough and facing the detection channel; A detector, and a supply unit for supplying the sample and the reagent to the microfluidic chip.

The first and second substrates may be formed of a material having a color capable of absorbing light or may be colored with a color capable of absorbing light. have.

The first substrate may be formed with a sample inlet for injecting a sample, a reducing agent inlet for injecting a reducing agent, a first reagent inlet for injecting the first reagent, and a second reagent inlet for injecting the second reagent .

Wherein the supply unit supplies the sample to the sample injection port, supplies the potassium sulfite to the reducing agent inlet, supplies the dissolved luminol to the first reagent injection port in the buffer solution of the basic condition, Hydrogen peroxide dissolved in the buffer solution can be supplied.

The supply unit may supply bromine ions to the microfluidic chip together with the first reagent and the second reagent, and the supply unit may include ethylenediaminetetraacetic acid (EDTA) together with the first reagent and the second reagent, To the microfluidic chip.

The detection channels are superposed and one end portion and the other end portion are alternately connected and may be formed so as to decrease from the center toward both side ends.

In the present invention, it is possible to make accurate analysis of chromium [III] and chromium [IV] by producing a reduction channel so that chromium [IV] in the sample is reduced to chromium [III] in the microfluidic chip.

By designing asymmetrically the reagent mixing channel injected with the chemiluminescent reagent and the reducing channel injected with the sample, the loss of chemiluminescence occurring in the reagent mixing channel is minimized, and chromium is detected with low detection limit and high sensitivity It is possible to do.

In addition, selective detection of chromium on the continuous stream is possible by removing the metal ions that can interfere with the chelating agent and the sample contained in the chemiluminescent reagent in the detection channel and present in the sample.

In addition, chromium [III] analysis does not cause precipitation of chromium [III] or coprecipitation with disturbing metals by injecting the sample into the reducing agent inlet, and it is possible to analyze the accurate chromium concentration.

In addition, since the slope of the calibration curve of chromium [III] is twice the slope of the calibration curve of total chromium, it is possible to analyze the concentrations of chromium [III] and chromium [IV] using only the calibration curve of total chromium, Can be reduced.

The microfluidic chip of this embodiment is made of a material that absorbs light to prevent ambient light from entering the detector, so that a stable chemiluminescent signal with high sensitivity can be obtained and it is easy to apply in the field.

1 is a plan view showing a top plate of a microfluidic chip according to an embodiment of the present invention.
2 is a plan view showing a bottom plate of a microfluidic chip according to an embodiment of the present invention.
3 is a configuration diagram illustrating a chrome detection apparatus according to an embodiment of the present invention.
FIG. 4A is a graph showing the chemiluminescent signal depending on the presence or absence of 0.1 M of bromine ion and the concentration of chromium [IV] in the chemiluminescent reagent. FIG. ], Respectively.
FIG. 5 is a graph showing a chemiluminescent signal according to the concentration of chromium [IV] obtained from a black microfluidic chip and a transparent microfluidic chip.
FIG. 6 is a photograph of the chemiluminescence of 500 ppb chromium [III] in a detection channel with a cooled CCD camera.
FIG. 7 is a graph showing the relationship between the concentration of the reagent mixture in the microfluidic chip (microfluidic chip A) and the length of the reagent mixing channel in the microfluidic chip (microfluidic chip B) Is a graph showing the chemiluminescent signal measured with standard samples in which the ratio of [III] to chromium [IV] is different.
8A is a graph showing a chemiluminescence signal according to the concentration of chromium [III] and chromium [IV] obtained by injecting a reducing agent, and FIG. 8B is a graph showing a calibration curve of chromium [III] and chromium [IV] Fig.
FIG. 9A is a graph showing chemiluminescence signals according to the concentrations of total chromium and chromium [III], and FIG. 9B is a graph showing calibration curves of total chromium and chromium [III].
10 is a graph showing the chemiluminescence signal of chromium [III] depending on the presence or absence of EDTA and the presence of a reducing agent in a sample mixed with chromium [III] and iron [II].
11 is a graph showing a chemiluminescence signal of chromium [III] obtained by injecting distilled water into a sample mixed with chromium [III] and iron [II].

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, which will be readily apparent to those skilled in the art to which the present invention pertains. The present invention may be embodied in many different forms and is not limited to the embodiments described herein.

FIG. 1 is a plan view showing a top plate of a microfluidic chip according to an embodiment of the present invention, and FIG. 2 is a plan view showing a bottom plate of a microfluidic chip according to an embodiment of the present invention.

The microfluidic chip 30 according to the present embodiment includes a top plate (first substrate) 10 and a bottom plate (second substrate) The upper plate 10 has a rectangular plate shape and the upper plate 10 has a sample inlet 11, a reducing agent inlet 12, a first reagent inlet 13 and a second reagent inlet 14. The sample injection port 11 is formed with a coupling part 31 for coupling with the sample injection tube 52. A coupling part 32 for coupling with the reducing agent injection tube 53 is formed in the reducing agent injection port 12, .

The first reagent injection port 13 is formed with a coupling part 34 for coupling with the reagent injection tube 54. The second reagent injection port 14 is provided with a coupling part 34 for coupling with the reagent injection tube 55, (35) are formed.

The upper plate 10 is provided with a sample channel 11a, a reducing agent channel 12a, a first reagent channel 13a, a second reagent channel 14a, a reducing channel 15, a reagent mixing channel 16, 17 are formed. A sample channel 11a is connected to the sample inlet 11 and a reducing agent channel 12a is connected to the reducing agent inlet 12. A sample containing chromium is injected through the sample inlet 11 and a reducing agent such as potassium sulfite is injected through the reducing agent inlet 12 to reduce chromium [IV].

A reducing channel 15 is connected to the sample channel 11a and the reducing agent channel 12a and the sample and the reducing agent are mixed in the reducing channel 15 and the chromium [IV] . The reduction channels (15) are superposed and one end and the other end are alternately connected. One end of the reduction channel 15 is connected to the sample channel 11a and the reducing agent channel 12a and the other end is connected to the detection channel 17. [

A first reagent channel 13a is connected to the first reagent inlet 13 and a second reagent channel 14a is connected to the second reagent inlet 14. [ The first reagent injection port 13 may be filled with luminol, bromine ion and EDTA dissolved in a buffer solution of a basic condition and may be injected into the buffer solution of the basic condition used for dissolving the luminol in the second reagent inlet 14 Dissolved hydrogen peroxide, bromine ions and EDTA can be injected.

A reagent mixing channel 16 is connected to the first reagent channel 13a and the second reagent channel 14a and a mixture of luminol and hydrogen peroxide is mixed in the reagent mixing channel 16. The reagent mixing channels 16 are superposed and one end and the other end are alternately connected. One end of the reagent mixing channel 16 is connected to the first reagent channel 13a and the second reagent channel 14a and the other end is connected to the detection channel 17. [

A reducing channel 15 and a reagent mixing channel 16 are connected to one end of the detection channel 17 and an outlet 18 is formed at the other end to discharge reagents and samples used in the reaction. The outlet (18) is formed with a fastening part (33) so that a discharge pipe can be coupled.

The detection channels 17 are superposed and one end and the other end are alternately connected. The length of the overlapping detection channels 17 decreases from the center to both side ends, and the center of the detection channel 17 is arranged to match the center of the circular detection portion 21. [

Since the reducing channel 15 and the reagent mixing channel 16 are connected together at one end of the detection channel 17, the sample and the reagent, which are mixed in the detection channel, are mixed to eliminate the interference effect in the sample and chemiluminescence reaction occurs.

The length of the reduction channel (15) is two to six times the length of the reagent mixing channel (16). As a result, the chromium (IV) can be reduced to chromium (III) by the reducing agent while passing through the reduction channel (15). In addition, since the length of the reagent mixing channel 16 is short, the loss of chemiluminescence occurring in the reagent mixing channel 16 can be minimized, and chromium can be detected with a low detection limit and a high sensitivity.

The fastening portions 31, 32, 33, 34, and 35 are made of polymer or tubing and have a hole so that the tube or tube can be easily attached and detached.

The upper plate 10 is made of a polymer having a light absorbing property, and may be made of black polydimethylsiloxane (PDMS). However, the top plate 10 may be made of a material having various colors having light absorbing properties, or may be colored with a color having light absorbing properties.

On the other hand, in the lower plate 20, a detecting portion 21 is formed at a lower portion corresponding to the detecting channel 17 of the upper plate 10. [ The lower plate 20 is made of black PDMS, and the detection unit 21 is filled with transparent PDMS so that the light generated by the chemiluminescence reaction can pass through. The detection unit 21 can be formed by forming a hole in a black PDMS and then injecting a transparent PDMS. However, when the lower plate 20 is colored, only the detection portion is formed without being colored.

As described above, when the upper plate 10 and the lower plate 20 are formed to have light absorbing properties, a stable chemiluminescence signal can be obtained by absorbing light incident from the outside, and the detection limit and sensitivity can be improved.

Hereinafter, a manufacturing method of the upper plate 10 and the lower plate 20 will be described. After the photoresist is spin-coated on the prepared silicon wafer, ultraviolet rays are irradiated while the photomask is provided, and then the positive and negative patterns are formed through curing and development.

When black PDMS is poured on a wafer having a bumpy pattern formed thereon, and the top plate 10 is separated from the wafer after curing, a black PDMS top plate with an engraved pattern can be obtained. The lower plate 20 is also manufactured in the same manner as the upper plate 10.

The center of the detection channel 17 of the upper plate 10 is coupled with the center of the detection portion 21 of the lower plate 20 so as to be coincident with the center of the detection channel 17 of the upper plate 10 after the upper plate 10 and the lower plate 20 are surface- do.

The sample and the reducing agent move to the detection channel 17 through the reducing channel 15 and the reagent moves to the detection channel 17 through the reagent mixing channel 16 to cause the chemiluminescence reaction. The sample and the reagent after the chemiluminescence reaction in the detection channel 17 move to the outlet 18.

3 is a configuration diagram illustrating a chrome detection apparatus according to an embodiment of the present invention.

3, the chrome detecting device according to the present embodiment includes a detector 42 and a microfluidic chip 30, which are inserted into a housing 41 forming an outer shape and disposed below the detecting unit 21, And a supply part 51 for supplying the sample and the reagent to the microfluidic chip 30. The housing 41 has a substantially rectangular box shape. The housing 41 is made of a metal colored in black so as to absorb light, and the microfluidic chip 30 is fixed to the upper surface of the housing 41. Accordingly, external light is prevented from entering the detector 42, and only the light generated by the chemiluminescence reaction is incident on the detector 42 through the detector 21.

A sample injection tube 52 is connected to the coupling part 31 provided at the sample injection port 11 and a reducing agent injection tube 53 is connected to the coupling part 32 provided at the reducing agent injection port 12. The first reagent injection tube 54 is connected to the coupling part 34 provided at the first reagent injection port 13 and the second reagent injection tube 34 is connected to the coupling part 35 provided at the first reagent injection port 14. [ (55) are connected and connected.

A peristaltic pump is installed in the supply part 51. The supply part 51 supplies the sample to the sample injection tube 52 and supplies potassium sulfite to the reducing agent injection tube 53. [ In addition, the supply part 51 supplies luminol to the first reagent injection tube 54 and hydrogen peroxide to the second reagent injection tube 55. [

The supply part 51 continuously supplies the reagent and the sample, and the supplied sample and the reagent are reacted and then discharged through the outlet 18. Accordingly, the chrome detection apparatus according to the present embodiment can continuously monitor chromium.

The supply part 51 supplies bromine ions to the microfluidic chip 30 together with the reagent, whereby the chemiluminescent intensity is increased to improve the sensitivity of the chromium detection device, and the chromium detection device has a lower detection limit. Further, the supply part 51 supplies EDTA together with the reagent. EDTA reacts with metal ions under basic conditions to form complexes, thereby eliminating other metal ions involved in the luminol chemiluminescence reaction contained in the sample, thereby preventing the chromium concentration from being measured incorrectly.

A power source 61 is connected to the detector 42. A digital multimeter 62 for processing the signal generated by the detector 42 is connected to the detector 42 and the digital multimeter 62 transmits the processed signal to the connected computer 63.

[Experimental Example 1]

In Experimental Example 1, the intensity of chemiluminescence according to the concentration of chromium was measured using the microfluidic chip and the chromium detection device including the microfluidic chip according to the present embodiment. The chemiluminescent reagent is for detecting chromium [III] in the sample and reacts with chromium [III] under basic conditions to exhibit chemiluminescence. The chemiluminescent reagent consists of luminol and hydrogen peroxide, and the intensity of chemiluminescence depends on the concentration of luminol and hydrogen peroxide.

The buffer solution for the chemiluminescence method was prepared by adding 30.09 g of boric acid (H ₃ BO ₃ ), 10.2 g of potassium bromide (KBr), 2.92 g of EDTA ((HOOCCH ₂ ) ₂ HNCH ₂ CH ₂ NH (CH ₂ COOH) ₂ ) Is dissolved in distilled water and adjusted to pH 10.9 with a 5 M potassium hydroxide (KOH) solution to give a final volume of 1 L. The chemiluminescent reagent is prepared by dissolving 0.02 g of luminol in a buffer solution to adjust the pH to 10.9 and a final volume of 100 mL. 0.868 mL of hydrogen peroxide as an oxidizing agent is dissolved in the buffer solution to adjust the pH to 10.9, and the final volume is adjusted to 100 mL. The reducing agent for reducing chromium [IV] to chromium [III] is prepared by dissolving 0.1104 g of potassium sulfite (K ₂ SO ₃ ) in distilled water to adjust the pH to 2.5, and then making the final volume to 100 mL. Standard samples of chromium [III] and chromium [IV] were prepared by dissolving chromium nitrate 9 hydrate (Cr (NO ₃ ) ₃ .9H ₂ O) and potassium chromate (K ₂ CrO ₄ ) The following dilutions are prepared at concentrations of 5 ppb, 50 ppb, 125 ppb, 250 ppb, 500 ppb and 1000 ppb, respectively.

The chemiluminescent reagent, luminol, is injected through the first reagent inlet 13 and hydrogen peroxide is injected through the second reagent inlet 14. The sample is injected into the sample inlet (11), and the reducing agent is injected through the reducing agent inlet (12). The flow rate of the peristaltic pump used when injecting each solution was 7.5 μl / min.

FIG. 4A is a graph showing the chemiluminescent signal depending on the presence or absence of 0.1 M of bromine ion and the concentration of chromium [IV] in the chemiluminescent reagent. FIG. ], Respectively.

The standard samples used in this experiment are 5 ppb, 50 ppb, 125 ppb, 250 ppb, 500 ppb and 1000 ppb chromium [Ⅳ], and a chemiluminescent reagent containing 0.1 M bromine ion and bromine ion The chemiluminescent signal was continuously measured according to each concentration by using a chemiluminescent reagent to which no reagent was added. Bromine ions attack complexes formed by the reaction of metal ions, hydrogen peroxide and luminol under basic conditions. As a result, 3-aminophthalate (3-aminophthalate), which causes chemiluminescence, is generated more and the intensity of chemiluminescence is increased. As shown in FIG. 4A, when the chemiluminescent reagent containing 0.1 M of bromine ion was used, the chemiluminescent intensity was greatly increased compared with the case of using the chemiluminescent reagent without the bromine ion.

In FIG. 4B, the calibration curve of the chemiluminescent reagent to which 0.1 M of bromine ion is added is y = 0.00100x + 0.00289 (R ² = 0.9999), and the calibration curve of the chemiluminescent reagent without bromine ion is y = 0.000182x + 0.000212 (R ² = 0.9999). Where y is the chemiluminescence intensity, x is the concentration, and R ² is the linear coefficient. These calibration curves show the relationship of the chemiluminescence intensity according to the concentration of the substance to be analyzed by measuring the chemiluminescence intensity from a low concentration to a concentration order using a standard sample prepared from the substance to be analyzed.

As shown in FIG. 4B, when the chemiluminescent reagent containing 0.1 M of bromine ion was used, the slope of the calibration curve increased about 5.5 times as compared with the case of using the chemiluminescent reagent without bromine ion addition.

When 0.1 M bromine ion is added, the detection limit is 0.32 ppb, which is about 4 times lower than the detection limit of 1.3 ppb without bromine ion addition. Therefore, it is possible to detect chromium with higher sensitivity and lower detection limit by adding 0.1 M bromine ion to the chemiluminescent reagent.

[Experimental Example 2]

In Experimental Example 2, the chemiluminescence intensity of total chromium was measured by applying both the upper plate 10 and the lower plate 20 of the microfluidic chip to black chromium PDMS and the transparent PDMS to the chromium detection apparatus. This experiment was carried out in a dark room.

FIG. 5 is a graph showing a chemiluminescent signal according to the concentration of chromium [IV] obtained from a black microfluidic chip and a transparent microfluidic chip.

As shown in FIG. 5, in the microfluid chip fabricated with transparent PDMS, the intensity of the background signal and the signal greatly increased due to the external light entering the detector, and the chromium [IV] standard sample (5 ppb, 50 ppb, 125 ppb, 250 ppb, 500 ppb, and 1000 ppb), the signal is unstable and the noise is large due to scattering of light generated in the ambient light and chemiluminescent reaction.

As the intensity of the background signal increases, chemiluminescent signals of chromium [Ⅲ] less than 50 ppb can not be distinguished from background signals. The chemiluminescence signal obtained from the microfluidic chip fabricated with transparent PDMS increases the detection limit according to the intensity of the background signal and the noise, and the chromium concentration range to be detected is reduced.

On the other hand, the microfluidic chip fabricated with black PDMS blocks external light from entering the detector, thus reducing the background signal intensity and noise, and the chemiluminescent signal is very stable. By using a microfluidic chip made of black PDMS, the chrome detection device effectively blocks external light, enabling detection of chrome with higher sensitivity and lower detection limit, and can extend the range of detectable chromium concentration have.

[Experimental Example 3]

In Experimental Example 3, 500 ppb chromium [III] was injected into the sample inlet and chemiluminescence was observed with a cooled CCD camera. FIG. 6 is a photograph of the chemiluminescence of 500 ppb chromium [III] in a detection channel with a cooled CCD camera. Chemiluminescence is most strongly expressed in the center portion of the detection channel 17, and the intensity of chemiluminescence at the beginning and end portions is relatively weak compared to the center portion. This is a result of showing that the design of the microfluidic chip and the condition of the reagent are optimized so that most of the light generated from the chemiluminescence reaction can be measured from the generation of chemiluminescence to the disappearance, and the chromium detection with high sensitivity is possible.

[Experimental Example 4]

In Experimental Example 4, in order to confirm the reduction ratio in which chromium [III] is reduced to chromium [IV], the total chromium concentration is made equal to 1000 ppb and the ratios of chromium [III] and chromium [ 5: 5, and 7: 3 were sequentially injected into the microfluidic chip A and the microfluidic chip B, respectively, to measure chemiluminescent signals.

FIG. 7 is a graph showing the relationship between the number of microfluidic chips A (reducing channel (15): 100 cm, reagent mixing channel (16): 20 cm) formed with a longer reductant channel than the reagent mixing channel, The ratio of chromium [III] to chromium [IV] in 1000 ppb total chromium in Fluid Chip B (Reducing Channel (15): 20 cm, Reagent Mixing Channel (20): 20 cm) Fig.

The microfluidic chip A shows the same chemiluminescence intensity even if the composition ratio of chromium [IV] and chromium [III] in the sample is different, because chromium [IV] contained in the sample is reduced to chromium [III].

In the microfluidic chip B, only a part of the chromium [IV] contained in the sample is reduced to chromium [III], and samples of different compositions show different chemiluminescent intensity.

As described above, according to the present invention, since the length of the reducing channel 15 is longer than the length of the reagent mixing channel 16, the concentration of chromium [IV] can be accurately measured.

[Experimental Example 5]

In Experimental Example 5, chromium [III] and chromium [III] were used in order to confirm that a wide concentration range of chromium [IV] was reduced to 100% by chromium [III] [IV] Each of the standard samples was injected into the sample inlet 11 sequentially, and a reducing agent was injected into the reducing agent inlet 12 to measure the chemiluminescence signal.

8A is a graph showing a chemiluminescence signal according to the concentration of chromium [III] and chromium [IV] obtained by injecting a reducing agent, and FIG. 8B is a graph showing a calibration curve of chromium [III] and chromium [IV] Fig.

In FIG. 8A, chromium [III] and chromium [IV] at each concentration show substantially the same chemiluminescence intensity.

In FIG. 8B, the calibration curve of chromium [III] is y = 0.00100x-0.00115 (R ² = 0.9999), and the calibration curve of chromium [IV] is y = 0.00100x + 0.00127 (R ² = 0.9999). Where y is the chemiluminescence intensity, x is the concentration, and R ² is the linear coefficient.

Since the calibration curves of chromium [III] and chromium [IV] have the same slope and the y-intercept is very different, each calibration curve is shown in one graph. As a result, two calibration curves overlap have.

From the results of FIG. 8, the microfluid chip and the chemiluminescence detection device including the same can 100% reduce chromium [IV] in a wide concentration range (5 to 1000 ppb) to chromium [III] ] And chromium [IV] can be accurately detected.

[Experimental Example 6]

In Experimental Example 6, chemiluminescence signals were measured according to the concentrations obtained from the total chromium and chromium [Ⅲ] standard samples of 5 ppb, 50 ppb, 125 ppb, 250 ppb, 500 ppb and 1000 ppb, respectively.

FIG. 9A is a graph showing chemiluminescence signals according to the concentrations of total chromium and chromium [III], and FIG. 9B is a graph showing calibration curves of total chromium and chromium [III].

It can be seen that the chromium [III] chemiluminescence intensity is twice that of the total chromium chemiluminescence intensity for a sample of the same concentration. As shown in FIG. 8A, when the reducing agent is injected, chromium [III] and chromium [IV] have almost the same chemiluminescence intensity at the same concentration. However, in Experimental Example 6, the sample is injected into the sample inlet 11 and the reducing agent inlet 12 during the chromium [III] analysis, and the sample is injected only into the sample inlet 11 and the reducing agent inlet 12, , The concentration of the sample is diluted to 1/2, and the chemiluminescence intensity of chromium [III] at the same concentration is twice the chemiluminescence intensity of total chromium.

FIG. 9B is a calibration curve obtained by repeating the chemiluminescence signal according to the concentration shown in FIG. 9A three times for each of the total chromium and chromium [III], and it can be seen that the relative standard deviation is very small because the reproducibility is very high. In FIG. 9B, the calibration curve of chromium [III] is y = 0.00200x + 0.00112 (R ² = 0.9999) and the calibration curve of total chromium is y = 0.00100x + 0.00289 (R ² = 0.9999). Where y is the chemiluminescence intensity, x is the concentration, and R ² is the linear coefficient.

According to Experimental Example 6, it can be seen that the slope of the chromium [III] calibration curve is twice the slope of the total chromium calibration curve. Using this relationship, the total chromium and chromium [Ⅲ] and chromium [Ⅳ] concentrations in the field sample were analyzed using the total chromium calibration curve and the results were calculated as two calibration curves (total chromium and chromium [Ⅲ] calibration curves) (Table 2). Thus, the chromium detection apparatus according to the present invention can accurately analyze the total chromium, chromium [III], and chromium [IV] using only the total chromium calibration curve, and can greatly reduce the analysis time.

The detection limits of total chromium and chromium [III] obtained from Experimental Example 6 are 0.32 ppb and 0.13 ppb, respectively. The linear range is from 5 ppb to 1000 ppb and contains legal regulated concentration. Therefore, it is possible to detect chromium without pretreatment such as sample dilution or concentration.

[Experimental Example 7]

The luminol chemiluminescence reaction also involves other metal ions (cobalt [II], iron [II], copper [II], nickel [II], etc.) in addition to chromium [III] The pH is highly dependent on pH. For example, due to the acidification of water caused by acid rain, etc., the pH of the water is reduced to 4.0 to 4.5, and the concentration of metal ions increases, so that the effect of the interfering metal ions becomes more serious. Thus, Experimental Example 7 confirms that chromium can be selectively detected without any disturbance by other metal ions even when the pH is changed due to acidification of water.

The effect of the interfering metal ions can be removed using EDTA. EDTA reacts with metal ions to form complexes under basic conditions, and complexed metal ions do not participate in chemiluminescence. On the other hand, chromium [III] forms a complex at a relatively slow rate compared to other metal ions when it reacts with EDTA, so that chromium can be selectively analyzed in the sample.

10 is a graph showing the chemiluminescence signal of chromium [III] depending on the presence or absence of EDTA and the presence of a reducing agent in a sample mixed with chromium [III] and iron [II]. 10 (a) is a chemiluminescence signal measured from a sample in which only 500 ppb of chromium [III] is dissolved. As a standard for the chemiluminescence intensity of other samples, the chemiluminescence intensity of (a) was set at 100%.

In FIG. 10 (b), a sample in which 500 ppb chromium [III] and 5 ppm iron [II] are dissolved is analyzed by luminol and hydrogen peroxide not containing EDTA, showing a chemical luminescence intensity of 117%. An increase in the chemiluminescence intensity of 17% is due to the inhibitory substance, 5 ppm iron [II].

10 (c) is a chemiluminescence signal obtained by injecting the same sample as in (b) and injecting a reagent in which 10 mM EDTA is dissolved in a buffer solution in which luminol and hydrogen peroxide are dissolved, respectively. Chemiluminescence intensity. From the above results, it was confirmed that chromium [III] could be selectively detected without interference with 5 ppm iron [II] using 10 mM EDTA.

In FIG. 10 (d), the chemiluminescence signal obtained by injecting the reducing agent into one of the two sample injection ports under the same experimental conditions as in (c) shows a chemiluminescence intensity corresponding to 50%. The chemiluminescence intensity of 50% is due to dilution of the concentration of the sample by ½ with the injection of the reducing agent. From the above results, it can be seen that the use of 10 mM EDTA does not affect the chemiluminescence signal even when a reducing agent having a pH of 2.5 is injected.

From the results of FIG. 10, it can be seen that chromium [III] and chromium [IV] can be selectively detected using EDTA without separation even when the concentration of disturbing metal ions is increased due to acidification.

[Experimental Example 8]

Experimental Example 8 was carried out in the same manner as in Example 1 except that 500 ppm of chromium [III] and 5 ppm of iron [II] were dissolved in the solution of pH 4.5 to prepare precipitates due to injection of distilled water during chromium [III] A solution of 500 ppb of chromium [III] and iron [II] dissolved in the sample inlet 11 is injected into the sample inlet 11 and the reducing agent inlet 12, and distilled water is injected into the reducing agent inlet 12 The chemiluminescent signal was confirmed.

In the prior art, distilled water (pH 5.8 to 6.2) was injected into one of two injection ports when chromium [III] was detected in the sample. In this case, during the mixing of the sample and the distilled water in the reducing channel 20, the pH of the sample may be changed and precipitation of chromium [III] may be caused.

11 is a graph showing a chemiluminescence signal of chromium [III] obtained by injecting distilled water into a sample mixed with chromium [III] and iron [II]. 11 (a) is a chemiluminescence signal obtained by injecting 500 ppb chromium [III] into the sample inlet 11 and the reducing agent inlet 12, and its chemiluminescence intensity is set at 100%. 11 (b) is a chemiluminescence signal obtained by injecting a sample in which 500 ppb chromium [III] and pH 5 iron [II] dissolved at pH 4.5 are injected into a sample inlet 11 and a reducing agent inlet 12, respectively, (a), which was 17% higher than that of (a). 11 (d), EDTA was added to the reagent to obtain a chemiluminescent signal. Thus, the increase in the chemiluminescence intensity of 17% in Figure 11 (b) results from the interference effect by 5 ppm iron [II].

11 (c) is a chemiluminescence signal obtained by injecting 500 ppb chromium [III] and 5 ppm iron [II] as a sample inlet and distilled water as a reducing agent inlet. If the sample is not affected by the injection of distilled water, the concentration of the sample should be diluted to 1/2 and show a chemiluminescence intensity of 58.5%. However, Fig. 11 (c) shows a chemiluminescence intensity of 17.3%. This is due to precipitation of chromium [III] or coprecipitation of iron and chromium due to the increase of pH after the sample of pH 4.5 mixed with distilled water in the reduction channel, resulting in a decrease of the chemiluminescence intensity by 41.2%. FIG. 11 (d) is a graph showing the results of a chemical emission signal obtained by injecting a sample and distilled water in the same manner as in FIG. 11 (c) and injecting a reagent dissolved with EDTA. As a result, iron [II] Which means that the chemiluminescence intensity of 5.4% is decreased. From the above results, when chromium [III] is detected by the chemiluminescence method, analysis error occurs when distilled water is injected. In this case, even if EDTA is injected, the interference effect by other metal ions can not be removed .

[Experimental Example 9]

In Experimental Example 9, iron [II], cobalt [II], copper [II], nickel [II] and the like participating in the luminol chemiluminescence reaction were reacted with a reagent to which EDTA was not added and 10 mM of EDTA Chemiluminescence of a sample with pH of 4.5 mixed with chromium was detected.

500 ppb of chromium [III] and 500 ppm of each of the interfering metal ions dissolved in 5 ppm were adjusted to pH 4.5 with nitric acid. At this time, the volume of the nitric acid solution used for adjusting the pH is 1/1000 or less of the volume of the sample, and the concentration change of the chromium and the interfering metal in the sample is negligible. And 500 ppb total chromium (including 250 ppb of chromium [Ⅲ] and 250 ppb of chromium [Ⅳ]) and disturbing metal ions of 500 ppb or 5 ppm dissolved in nitric acid to adjust the pH to 4.5 Respectively.

The chemiluminescent reagent was prepared by adding 10 mM EDTA buffer solution and laminol and hydrogen peroxide to the buffer solution without EDTA.

[Table 1]

In Table 1, the chemical luminescence intensity of 500 ppb chromium [III] and 500 ppb chromium [III] sample are set at 100%, and 500 ppb total chromium and interfering metal ions are dissolved In the case of the sample, when the chemiluminescence intensity of 500 ppb total chromium sample was 100%, and the chemiluminescence reagent without EDTA and 10 mM EDTA were injected, the chemiluminescence intensity of each sample was measured . The value in parentheses next to the chemiluminescence intensity is the relative standard deviation obtained by measuring the chemiluminescence intensity for each sample three times.

When a reagent without EDTA was added, the intensity of chemiluminescence measured by the interfering metal ions in the sample exceeded 100%. However, in the case of using a chemiluminescent reagent containing 10 mM of EDTA, the disturbing metal ion was removed by EDTA in the detection channel, so that the same chemiluminescent intensity as 500 ppb of chromium [III] and 500 ppb of total chromium standard sample And 500 ppb of chromium [III] and total chromium can be selectively analyzed without disturbing effect even for samples with a concentration of disturbing metal ions of 10 times (5 ppm).

[Experimental Example 10]

In Experimental Example 10, the concentrations of total chromium, chromium [III] and chromium [IV] were analyzed in a sample collected at a factory to confirm the performance of the detection apparatus according to the present embodiment. The chromium concentration was measured by injecting a sample with a filter of 0.45 μm pore into the microfluidic chip.

Table 2 compares the concentrations of total chromium, chromium [III] and chromium [IV] in the sample and the results of the atomic absorption spectrophotometry and the absorption spectrometry. The atomic absorption spectrophotometry can only analyze the total chromium concentration , And the detection limit is 20 ppb. The absorption spectrum of the red complex produced by the reaction of chromium [Ⅳ] with 1,5-diphenylcarbazide is measured by ultraviolet-visible spectrophotometer at 540 nm wavelength. , And it has high selectivity for chromium [IV] analysis because there is no interference effect by other substances.

The chromium in the sample according to Experimental Example 10 was analyzed by the chemiluminescence method using the microfluidic chip according to the present embodiment. As a result, the concentrations of total chromium contained in Sample 1, Sample 2 and Sample 3 were 137 ppb and 112 ppb , And 128 ppb, respectively. As a result of triplicate analysis for each sample, the relative standard deviation was lower than that of atomic absorption spectrophotometry.

[Table 2]

The concentration of chromium [Ⅲ] analyzed by chemiluminescence method was measured by the atomic absorption spectrophotometry of 27 ppb, 20 ppb and 28 ppb of sample 1, sample 2 and sample 3, respectively. Ⅲ] concentration calculated by subtracting the concentration of chromium [Ⅲ]. The concentration of chromium [Ⅳ] was confirmed to be similar to that of chromium [Ⅳ], which was calculated by using the concentration of total chromium and chromium [III] and by the absorption method. Thus, it can be seen that the concentrations of total chromium, chromium [III], and chromium [IV] analyzed using the chrome detection apparatus according to this embodiment were measured accurately.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Of course.

10: upper plate 11, 12: sample inlet
13, 14: Reagent inlet 11a, 12a: Sample channel
13a, 14a: Reagent channel 15: Reduction channel
16: reagent mixing channel 17: detection channel
18: Solution outlet 20: Lower plate
21: detecting section 30: microfluidic chip
31, 32, 33, 34, 35: fastening portion 41: housing
42: detector 51:
52, 53: sample injection tube 54, 55: reagent injection tube
61: power supply 62: digital multimeter
63: Computer

Claims (15)

A reagent mixing channel for mixing a reagent and a reducing channel for mixing a sample and a reagent; and a first substrate having a reducing channel and a reagent mixing channel connected in parallel at one end and a detection channel connected to an outlet at the other end; And
And a second substrate coupled to the first substrate, the second substrate having a detection portion formed to face the detection channel and transmitting light therethrough,
Wherein the first substrate and the second substrate are made of a material having a color capable of absorbing light or colored with a color capable of absorbing light. The method according to claim 1,
Wherein the reducing channel is longer than the reagent mixing channel. delete 3. The method of claim 2,
A sample inlet for injecting a sample and a reducing agent inlet for injecting a reducing agent are formed on the first substrate, the sample inlet is connected to a reducing channel via a sample channel, and the reducing agent inlet is connected to a reducing channel And a microfluidic chip connected thereto. 5. The method of claim 4,
A first reagent injection port for injecting the first reagent and a second reagent injection port for injecting the second reagent are formed on the first substrate and the first reagent injection port is connected to the reagent mixing channel through the first reagent channel, And the second reagent inlet is connected to the reagent mixing channel through the second reagent channel. 3. The method of claim 2,
Wherein the detection channels are superposed and one end and the other end are alternately connected, and the microchip chip is formed so as to decrease from the center toward both ends. 3. The method of claim 2,
Wherein the reducing channel and the reagent mixing channel are overlapped and one end and the other end are alternately connected. A reagent mixing channel for mixing a sample and a reagent and a reagent mixing channel for mixing the reagent and a first substrate having the reduction channel and the reagent mixing channel connected in parallel at one end and the detection channel connected to the outlet at the other end, A microfluidic chip including a second substrate coupled to the first substrate, the microfluidic chip having a detection unit facing the detection channel and configured to transmit light;
A detector arranged to face the detector; And
A supply part for supplying a sample and a reagent to the microfluid chip;
/ RTI >
Wherein the first substrate and the second substrate are made of a material having a color capable of absorbing light or colored with a color capable of absorbing light. 9. The method of claim 8,
Wherein the reducing channel is longer than the reagent mixing channel. delete 10. The method of claim 9,
The first substrate is provided with a chromium detection device having a sample inlet for injecting a sample, a reducing agent inlet for injecting a reducing agent, a first reagent inlet for injecting the first reagent, and a second reagent inlet for injecting the second reagent, . 12. The method of claim 11,
Wherein the supply unit supplies the sample to the sample injection port, supplies the potassium sulfite to the reducing agent inlet, supplies the dissolved luminol to the first reagent injection port in the buffer solution of the basic condition, A chromium detection device for supplying hydrogen peroxide dissolved in a buffer solution. 13. The method of claim 12,
And the supply unit supplies bromine ions to the microfluidic chip together with the first reagent and the second reagent. 13. The method of claim 12,
Wherein the supply unit supplies ethylenediaminetetraacetic acid (EDTA) together with the first reagent and the second reagent to the microfluidic chip. 10. The method of claim 9,
Wherein the detection channels are superposed and one end portion and the other end portion are alternately connected to each other, and the chrome detection device is configured to decrease from the center to both side ends.

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