KR20050029620A - Method for detecting endotoxin using lap-on-a-chip - Google Patents

Abstract

Provided is a method for detecting endotoxin to analyze quantitatively endotoxin precisely in a short time with a small amount of sample by using a lap-on-a-chip. The method comprises the steps of injecting a control standard endotoxin sample containing a known concentration of endotoxin and a reagent for LAL (Limulus Amebocyte Lysate) test through the sample injection hole of a lap-on-a-chip comprising a replica which has a channel where a sample flows formed at the lower part of the replica, a sample injection hole formed at the one side terminal of the channel and a color detection part capable of the color change of sample formed at the other terminal, and a glass substrate which is combined with the lower part of the replica to form a channel and covers the channel; measuring the time which the absorbance of the sample of the color detection part reaches a certain value to obtain a standard curve showing the relationship between the concentration of endotoxin and the time reaching a certain absorbance value; injecting the sample whose endotoxin content is to be measured and the reagent for LAL test through the sample injection hole; and measuring the time which the absorbance of the sample of the color detection part reaches a certain absorbance value and putting the measured time value in the standard curve to obtain the content of endotoxin in the sample.

Description

Method for detecting endotoxin using lap-on-a-chip}

The present invention relates to a method for detecting endotoxin using a lap-on-a-chip, and more particularly, to quantitatively analyze endotoxin in a short time by using a small amount of samples. The present invention relates to a method for detecting endotoxin using a lab-on-a-chip.

Recently, research on analytical technologies using microfluidics chips has been actively conducted in the field of biotechnology, and the analytical methods using such microfluidic devices allow various diagnostics and tests to be quickly performed with only a small amount of sample. It has the advantage of being able to perform, making it a popular next generation analysis system. Microfluidic devices are mainly manufactured by applying a semiconductor fabrication process. Representative examples of such processes include surface micromachining and bulk micromachining, and such a method includes batch processing of microfluidic devices. It is useful in that mass production is possible. Initially, microfluidic devices were manufactured using silicon substrates commonly used in semiconductor manufacturing processes, but since silicon substrates are opaque and not suitable for biochemical samples, a method of manufacturing microfluidic devices using glass or polymer materials is currently available. It is proposed. Glass has the advantages of being less reactive and transparent with biochemical samples and having properties similar to those of silicon. However, glass has the disadvantages of being difficult to process and expensive materials and processing costs, such as REM (replica molding), μTM (micro transfer molding) and MIMIC. Research into a method for manufacturing a microfluidic device using a polymer material that can be mass-produced by micro molding in capliaries and hot embossing has been actively conducted. A lab-on-a-chip (LOC) manufactured using such a microfluidic device is a device that can continuously perform sample pretreatment, reaction, detection, and analysis on a chip. In this case, the analysis time is shortened to a few minutes or a few seconds, the amount of the sample used can be reduced to less than or less and there is an advantage that can be simultaneously analyzed for multiple samples.

Endotoxin, on the other hand, generally exists in the outer wall of Gram-negative bacteria and is not expressed while the bacteria are alive, but is released to the outside as the cell wall breaks when the bacteria proliferate or die. This endotoxin is combined with LAL (Limulus Amebocyte Lysate), a circulating blood cell extract of Limulus polyphemus , distributed on the Atlantic coast of the Americas to form a gel. As such, the method of detecting or quantifying endotoxin based on a reaction in which endotoxin activates LAL and causes gelation is called an LAL test method. Mainly used to LAL assays for indexing gel formation to detect or quantify endotoxins include gel clot, kinetic turbidimetric assay (KTA), or kinetic chromogenic assay (KCA), as shown in Scheme 1 below. As described above, the gelation method is sequentially activated by various endotoxin-reducing coagulation factors present in the LAL, and coagulogen is converted into coagulin by a finally activated coagulation enzyme to form a gel. Reaction (a) is used, and the turbidity method is a method of measuring the turbidity change generated in the reaction (a) in which the coagogen is gelled with coaglin, using an optical analyzer at 340 nm. Instead, using synthetic chromogenic peptide (chromogenic peptide) and measuring the degree of color development at 405 nm after incubation for a predetermined time, the result of the enzyme reaction activity Can be confirmed by release of developmental substrate and formation of yellow color (reaction b), and the degree of color development is proportional to the amount of endotoxin. In Scheme 1 below, the pNA substrate represents Boc-Leu-Gly-Arg-CONH-para-nitroaniline.

However, such a conventional endotoxin test method has a disadvantage of requiring a reaction time of about 1 hour and a large amount of a sample of about 100 μL of LAL solution with respect to 100 μl of the sample. Therefore, endotoxin testing of rare samples such as therapeutic proteins is not required. There is a problem that is not appropriate.

Accordingly, an object of the present invention is to provide a method for detecting endotoxin using a lab-on-a-chip capable of accurately detecting and quantifying endotoxin in a short time while using a small amount of a sample. Still another object of the present invention is to provide a method for efficiently detecting endotoxin using a lab-on-a-chip capable of economically mass production.

In order to achieve the above object, the present invention is a channel through which a sample flows is formed, a sample inlet is formed at one end of the channel, the other end of the color to measure the change in the color of the sample inside the channel It contains an endotoxin of a known concentration through the sample inlet of the lab-on-a-chip comprising a glass substrate covering the channel and coupled to the bottom of the replicator to form a flow path through which the detection unit and the replicator are formed. Injecting a control standard endotoxin sample and a reagent for testing Limulus Amebocyte Lysate (LAL); Measuring a time at which the sample of the color detection unit reaches a predetermined absorbance value and preparing a standard curve indicating a relationship between the concentration of endotoxin and the time at which the predetermined absorbance value is reached; Injecting a sample to measure the content of endotoxin and the reagent for LAL test into the lab-on-a-chip through the sample inlet; And measuring the time at which the sample of the color detection unit reaches a predetermined absorbance value and substituting the measured time value into the standard curve to quantify the endotoxin content in the sample. To provide.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1A and 1B are respectively a plan view and a cross-sectional view of a lab-on-a-chip that can be used in the method for detecting endotoxin according to the present invention. As shown in Figure 1a and 1b, the lab-on-a-chip 10 that can be used in the present invention is formed with a channel (channel, 14) through which the sample flows, the sample at one end of the channel (14) The injection port 12 is formed, and at the other end, a replicator 22 and a flow path through which the sample flows are formed, in which a color detector 16 capable of measuring a change in the sample color inside the channel 14 is formed. It is coupled to the bottom of the replicator 22 to form a glass substrate 20 covering the channel (14). In addition, the sample inlet 12 is equipped with a sample injection tube 24 to facilitate the injection of the sample, the color detection unit 16 is a color for easily detecting the color of the sample in the channel 14 The observation tube 26 may be further mounted. The replicator 22 is made of a plastic material such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polypropylene (PP), polystyrene (PS), and the like. In the lab-on-a-chip 10 that can be used in the method for detecting endotoxin according to the present invention, it is preferable to use polydimethylsiloxane. In this way, the use of polydimethylsiloxane (PMDS) is not only advantageous for observing the color change of the sample present in the channel 14, but also for mass production, and has the advantage of easily forming various channels 14. have. As described above, PDMS is not toxic, easy to process, and inexpensive. However, since PDMS itself absorbs UV, it is difficult to accurately measure changes in absorbance. The light transmittance at 405 nm according to the thickness of the PDMS was measured and the results are shown in FIG. 2. From FIG. 2, when the thickness of the PDMS is 4 mm or less, the transmittance is 80% or more, and the thinner the thickness, the higher the transmittance. Therefore, the replicator 22 may be manufactured to have a thickness of about 1 to 4 mm, and if the thickness of the replicator 22 is less than 1 mm, the replicator 22 may be easily bent or broken. When the thickness of the replicator 22 exceeds 4 mm, the transmittance of light is so low that it is difficult to observe the color change of the sample in the channel 14. The size of the lab-on-a-chip 10 that may be used in the present invention may vary depending on the volume of the endotoxin sample used, but may have a length of about 4 to 8 mm and a width of about 10 to 30 mm. ) May have a width of about 1 to 3 mm, a length of 20 to 60 mm, and a depth of 50 to 200 μm.

The replicator 22 of the lab-on-a-chip 10 used in the present invention is manufactured by applying a photolithography process used in semiconductor manufacturing, and the manufacturing process of the replicator 22 is shown in FIG. 3. Shown. In order to manufacture the replicator 22, first, as shown in FIG. 3, a negative-photoresist is coated on the silicon substrate 30 to a predetermined thickness, preferably about 100 to 300 mu m. Then, the solvent contained in the coated photoresist is removed by heating at about 60 to 150 ° C. for 10 minutes to 1 hour to form a photoresist layer 32 (step a). After forming the photoresist layer 32 as described above, the photoresist layer 32 is etched into a predetermined pattern by using a quartz mask, a film mask, or the like, which is generally used in a photolithography process. (mold) is prepared (step b). Next, a polymer such as PDMS and a curing agent are mixed in a mold by weight ratio of about 10: 1 and poured into a mold, and then the mold is placed in a vacuum oven and maintained in a vacuum state for 1 hour to generate PDMS and the curing agent. The bubbles are removed and heated to cure (step c). The cured PDMS replicator 22 was removed from the oven and separated from the mold (step d), and the sample inlet 14 was formed by using a punch or the like equipment (step e), followed by a reactive ion etching (RIE) device. After treating the surface of the replicator 22 with an O ₂ plasma at, it may be bonded to the glass 20 to prepare a lab-on-a-chip 10 which may be used in the method for detecting endotoxin according to the present invention (step f). ). At this time, the sample injection tube 24 may be inserted into and fixed to the sample inlet 14 formed in the replicator 22 as necessary.

Through the sample inlet 14 of the lab-on-a-chip 10 prepared as described above, a control standard endotoxin (CSE) containing a known concentration of endotoxin (CSE) and a reagent for testing Limulus Amebocyte Lysate (LAL) are injected. The color detection unit 16 measures the time to reach a predetermined absorbance value and creates a standard curve. In this case, the concentration of the LAL test reagent may be set in a wide range in consideration of the flowability and color development of the reagent, but preferably has a concentration of about 30 to 50 mg / ml, and the LAL test reagent and the control standard endotoxin sample are the same. It is preferable to use by volume ratio. When the standard curve is prepared as described above, the sample to measure the endotoxin content and the reagent for testing Limulus Amebocyte Lysate (LAL) are injected into the lab-on-a-chip 10 through the sample inlet 14, and the color detection unit 16 determines the predetermined amount. After measuring the time to reach the absorbance value, the endotoxin content in the sample can be quantified by substituting the measured time value into the standard curve.

When using a lab-on-a-chip according to the present invention and quantitatively analyzing endotoxins by colorimetry, high sensitivity is shown at low volumes and the same absorbance at the same endotoxin concentration regardless of the volume of the sample. In addition, when the endotoxin is quantitatively analyzed by the kinetic point method for measuring the time to reach a predetermined absorbance value according to the present invention, the linear correlation coefficient of the obtained standard curve is 0.98 or more, which enables highly precise endotoxin content analysis. It can be seen that the time required for the measurement can be shortened within 10-15 minutes, and the volume of the sample for analyzing the endotoxin content can be reduced to 10 μl or less. In addition, the endotoxin detection method according to the present invention has the advantage of ensuring the reproducibility of endotoxin detection because it uses a lab-on-a-chip capable of mass production in the same form by a mold.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by the following Examples.

Materials and methods

Limulus Amebocyte Lysate (LAL) test reagent contains control standard endotoxin (CSE) to produce a standard curve of LAL that reacts with endotoxin to form a gel, and an endotoxin of 0.001 EU / ml or less, and dissolves CSE and LAL. It is composed of LRW (LAL reagent water), and all the vitreous and instruments used in the test were endotoxin-free products. LAL tests were carried out using the non-tax method and the colorimetric method, and all reagents for this were purchased from Endosafe, USA. All tests were performed at 37 ± 1 ° C and ELISA readers (ELISA reader, Power Wave x 340, BIO-TEC Instruments Inc., USA) and 96 well plates were used to measure turbidity and colorimetric changes. .

Endotoxin measuring method

In order to measure LAL, 100 μl of the LAL reagent and the endotoxin sample were mixed, and then reacted without stirring at 37 ± 1 ° C. for 1 hour to observe gel formation. In order to ensure the accuracy and effectiveness of this LAL test method, the labeling sensitivity of the LAL reagent was confirmed prior to the limit test method, and it was confirmed that the specimen did not promote or inhibit the reaction. The limit test consisted of negative control and positive control, and each test was repeated twice. Negative control is a test to determine whether the LRW used to dilute the sample solution or make an endotoxin standard with LAL reagent, and positive control is a test to confirm the reaction interference factor of the negative control and the sensitivity of the reagent. It was tested with an endotoxin standard solution corresponding to twice the sensitivity.

After the limit test, a standard curve for endotoxin was made using CSE of 0.05, 0.1, 0.5, 1, 2, 10 EU / ml. The LAL test method for color quantitative analysis was performed using the colorimetric method and the turbidity method. The end point method and the kinetic point method were tested when preparing a standard curve. The end point method is a method of measuring the color development according to the concentration of CSE at 405 nm after a predetermined time elapses, and the kinetic point method reaches a constant optical density at 405 nm. How to measure the time.

Determine how LAL is measured

To find out which of the turbidity and colorimetric methods used for LAL measurements were suitable for use on the chip, each method was compared at low CSE concentrations and small sample volumes. In the experiment, the gelation method was excluded because the chip could not be used to invert the reactant to determine whether the reactant flowed down. On-chip measurements require low concentrations of endotoxin detection at very low volumes, thus measuring the degree of change at a CSE concentration of 0.05-0.06 EU / ml and a volume of 50 μl. In the first 5 minutes, the change in color turbidity or colorimetric method was similar, but after 10 minutes, the color development of the colorimetric method rapidly increased. After 60 minutes, the colorimetric method showed absorbance of 0.05 at 340 nm, but the colorimetry showed 0.52 at 405 nm. . Therefore, it can be seen that the colorimetric method is about 10 times higher sensitivity than the turbidity method for low concentration of CSE and small volume measurement method.

Effect of sample volume on colorimetric method

In order to implement the LAL test on the lab-on-a-chip, we confirmed the effect of sample volume reduction on the measurement method. While decreasing the volume of the 0.05EU / ml CSE solution from 200 μl to 50 μl, the absorbance at 405 nm (Optical Density: OD) was measured, the results are shown in Figure 4a. In FIG. 4A, ■ indicates absorbance when the volume of the sample is 50 µl, ▲ indicates absorbance when the volume of the sample is 100 µl, and indicates the absorbance when the volume of the sample is 200 µl. From FIG. 4a, it can be seen that the same behavior is observed until the volume of the sample is 50 μl, and the gelation reaction is completed after all 50 minutes. The absorbance value also decreased as the volume was reduced. This is because the diameter of each well of the 96-well plate, which is the measuring instrument, is constant, but the volume is decreased and the height of the sample is actually reduced. In order to confirm this, the measured absorbance value was divided by the height of the sample, and the absorbance was calculated for the unit height, which is shown in Table 1 below.

Sample volume (μl) OD _405nm (-) Sample height (mm) OD / sample height (mm ^-1 ) 50 0.523 1.0 0.52 100 1.123 2.5 0.45 200 2.404 5.0 0.48

From Table 1, it can be seen that the absorbance has a similar value regardless of volume. That is, it can be seen that the same LAL reaction occurs regardless of the volume at the same CSE concentration, and it is important to keep the depth of penetration constant regardless of the volume of the sample by adjusting the flow path during on-chip measurement. It can be seen.

Effect of sample concentration on colorimetric method

The volume of the CSE sample was fixed at 50 μl, and the optical density at 405 nm was measured while changing the concentration of the CSE sample to 0.05, 0.5, and 5 EU / ml. The results are shown in FIG. 4B. . 4b is the absorbance when the concentration of the CSE sample is 0.05 EU / ml, the ▲ is the absorbance when the concentration of the CSE sample is 0.5 EU / ml, and the case where the concentration of the CSE sample is 5 EU / ml. The absorbance of is shown. 4b, it can be seen that the change in absorbance value according to the concentration of CSE shows a large difference in the beginning but similar values after 50 minutes.

Based on these results, endotoxin quantitative standard curves were prepared using an end point method and a kinetic point method, respectively. The end point method measured the absorbance values at 20 minutes to clearly distinguish the amount of change according to the concentration of each CSE, and the results are shown in FIG. 5A. In the kinetic point method, 0.1 absorbance was measured. The time indicating the change was measured and the result is shown in FIG. 5B. In FIG. 5A and 5B, * indicates absorbance when the sample volume is 50 µL, ▲ indicates absorbance when the sample volume is 100 µL, and indicates the absorbance when the sample volume is 200 µL. 5A and 5B, the linear correlation coefficient (R ² ) is inferior to 0.84 in average in the end point method with fixed measurement time, but the linear correlation coefficient is more than 0.99 in average in the kinetic point method with fixed absorbance. You can see this is very high. Therefore, according to the standards of the Korea Food and Drug Administration, it can be seen that the kinetic point method whose linear correlation coefficient (r ² ) of the standard curve satisfies 0.98 or more is more useful for the detection of endotoxin using Lab-on-a-Chip than the endpoint method.

PDMS Lab-on-a-Chip (LOC)

To fabricate PDMS lab-on-a-chip, first spin-coating a negative photoresist SU-8 (NANO ™, SU-8, MICROCHEM, USA) to a thickness of 100 μm on a silicon substrate and then heating at 65 ° C. for 10 minutes. After heating at 96 ° C. for 30 minutes to remove the solvent contained in the coated photoresist, a structure (mold) was manufactured using a photolithography process. As a mask for manufacturing such a mold, a quartz mask and an inexpensive film mask, which are generally used in a photolithography process, are used. Next, PDMS (SYLGARD184, Dow Corning, USA) was mixed with a curing agent in a weight ratio of 10: 1 to pour the mold into the mold, and the mold was placed in a vacuum oven and kept in vacuum for 1 hour. By removing the bubbles generated during the mixing process of PDMS and the curing agent, the mixture was cured by heating at 65 ℃ for 4 hours in a thermo-hygrostat (MTH2200, SANYO, Japan). The cured PDMS replicator is taken out of the oven, separated from the mold, formed a hole for tubing with a punch, and then the surface of the mimetic with O ₂ plasma in a reactive ion etching (RIE) instrument. After the treatment, the glass was bonded to Pyrex slide glass, a sample injection tube was inserted and fixed, and a PDMS microfluidic device was manufactured.

An example of a mold for mass production of such PDMS lab-on-a-chip is shown in FIG. 6a, and an example of a lab-on-a-chip for LAL measurement is shown in FIG. 6b. The chip shown in FIG. 6B has a straight shape, and includes a portion capable of injecting a sample in front and a portion capable of measuring color change in the rear portion. The length of the straight chip was 62 mm and the width was 18 mm. The width, length, and depth of the flow path were 2 mm, 44.34 mm, and 100 μm, respectively, and the thickness of the manufactured PDMS replicator was 2 mm.

LAL test using PDMS LOC

LAL experiments were performed using LOC, and the change in color development with time was measured by colorimetric method for CSE concentrations of 0.05, 0.5, and 5 EU / ml. The results are shown in FIG. 7. In FIG. 7 is the absorbance when the concentration of the CSE sample is 5 EU / ml, the ▲ is the absorbance when the concentration of the CSE sample is 0.5 EU / ml, and the case where the concentration of the CSE sample is 0.05 EU / ml. The absorbance of is shown. As shown in Figure 7, it can be seen that as the endotoxin concentration increases, the change in the initial absorbance is severe and becomes constant or rather decreases after 10 minutes. In addition, by using the LOC, a kinetic point method for measuring the time when the absorbance value at 405nm reaches 0.01 is prepared in the standard curve for each CSE concentration is shown in FIG. As shown in FIG. 8, the time to reach the endotoxin concentration and the predetermined absorbance was 0.99 or more, and the time required for the measurement was also shortened to within 10-15 minutes.

From the above results, the conventional LAL test method was able to obtain a result after the reaction for one hour at 37 ℃, using the lab-on-a-chip method according to the present invention can not only shorten the measurement time within 10 minutes, but also measured It can be seen that the volume of the sample used for can be reduced from 100 μl to 4.43 μl. In addition, the endotoxin detection method using the lab-on-a-chip according to the present invention can be seen that the accuracy of the detection is very high compared to the conventional method.

As described above, the method for detecting endotoxin using the lab-on-a-chip according to the present invention can not only accurately detect and quantify endotoxin in a short time while using a small amount of sample, but also can be economically mass-produced. By using on-chip, endotoxin can be efficiently detected with reproducibility.

1A and 1B show a plan view and a cross-sectional view of a lab-on-a-chip that can be used in the method for detecting endotoxin according to the present invention, respectively.

Figure 2 is a graph showing the results of measuring the light transmittance at 405nm according to the thickness of the polydimethylsiloxane.

3 is a view for explaining a manufacturing process of the lab-on-a-chip used in the present invention.

4A and 4B are graphs showing the results of measuring absorbance at 405 nm by performing LAL test while varying the volume and concentration of the control standard endotoxin sample, respectively.

Figures 5a and 5b is a graph showing the endotoxin quantitative standard curve made using the end point (kinetic point) method, respectively.

6a and 6b are photographs showing an example of a mold and a lab-on-a-chip for mass production of the lab-on-a-chip used in the present invention, respectively.

Figure 7 is a graph showing the results of the LAL experiment by colorimetric method using a lab-on-a-chip while changing the concentration of the control standard endotoxin sample.

Fig. 8 is a graph showing a standard curve created by measuring the time at which the absorbance value at 405 nm reaches a predetermined value using a lab-on-a-chip while varying the concentration of the control standard endotoxin sample.

Claims (4)

The sampler has a channel through which a sample can flow, a sample inlet is formed at one end of the channel, and a replicator and a sample having a color detector for measuring a change in the sample color inside the channel. A control standard endotoxin sample containing a known concentration of endotoxin and LAL (Limulus Amebocyte) through a sample inlet of a lab-on-a-chip comprising a glass substrate covering the channel and coupled to the bottom of the replicator to form a flow path that can flow. Lysate) injecting a test reagent; Measuring a time at which the sample of the color detection unit reaches a predetermined absorbance value and preparing a standard curve indicating a relationship between the concentration of endotoxin and the time at which the predetermined absorbance value is reached; Injecting a sample to measure the content of endotoxin and the reagent for LAL test into the lab-on-a-chip through the sample inlet; And And measuring the time when the sample of the color detection unit reaches a predetermined absorbance value and substituting the measured time value into the standard curve to quantify the endotoxin content in the sample. According to claim 1, The sample inlet is equipped with a sample injection tube for facilitating the injection of the sample, the color detector is further equipped with a color observation tube for detecting the color of the sample in the channel Endotoxin detection method using on-chip. The method of claim 1, wherein the replicator is made of polydimethylsiloxane. The method of claim 1, wherein the linear correlation coefficient of the standard curve is 0.98 or more.