KR20110021570A - Microfluidic chip comprising thermoresponsive fluorogenic conjugated polymer as a temperature sensor and temperature measurement method in micro channel of the microfluidic chip - Google Patents

Abstract

PURPOSE: A microfluidic chip with a thermoresponsive fluorogenic conjugated polymer as a temperature sensor and a temperature measurement method of a micro channel and a microfluidic chip are provided to measure temperature by only observing the intensity of fluorescence. CONSTITUTION: A temperature measurement method of a micro channel and a microfluidic chip is as follows. A thermoresponsive fluorogenic conjugated polymer is used as a temperature sensor. The thermoresponsive fluorogenic conjugated polymer is injected into a micro channel(130) of the microfluidic chip. The fluorescence intensity of the thermo-reception fluorescence conjugated polymer generated by the temperature of the micro channel is measured. The temperature of the micro channel is measured by measuring the fluorescence intensity of the microfluidic chip.

Description

Microfluidic chip containing temperature-sensitive fluorescent conjugated polymer as temperature sensor and temperature measurement method of microchannel in microfluidic chip

The present invention relates to a microfluidic chip comprising a temperature sensitive fluorescent conjugated polymer as a temperature sensor and a method for measuring a temperature of a microchannel in a microfluidic chip using a temperature sensitive fluorescent conjugated polymer as a temperature sensor. A microfluidic chip comprising a temperature-sensitive fluorescent conjugated polymer as a temperature sensor and a method of measuring the temperature of a microchannel in a microfluidic chip using a temperature-sensitive fluorescent conjugated polymer as a temperature sensor using a fluorescence property when the polymer is exposed to heat. will be.

Microfluidic chips, expressed in various terms such as Lap-on-a-chip, MEMS, Micro TAS, microfluidic chips, and nanofluidic chips, can be used to refine microbial and chemical laboratory components. Implementing means that experiments can be performed on one chip. That is, all of the processes such as sample pretreatment, transportation, control, and analysis take place on one chip.

These microfluidic chips are rapidly developing due to the advantages of ultra-fast analysis, minimizing the consumption of samples and reagents, simultaneous analysis of multiple samples, portable, low cost, and mass production. .

To be used as a microfluidic chip, low power consumption and shorter analysis time for real-time diagnosis are required for portable batteries. To this end, it is necessary to design and fabricate a structure that is thermally isolated but has a low thermal mass.

For example, DNA microfluidic chips require a lot of heating in the process of DNA processing, in particular DNA amplification, reaction control, or fluid, which is mainly used in cell lysis, polymerase chain reaction (PCR). The work of heating heat to about 40-100 degreeC in conveyance etc. is calculated | required.

In order to apply the microfluidic chip to such a task, accurate temperature measurement inside the microfluidic chip is required.

Meanwhile, polydiacetylene is a polymer of diacetylene monomer, and is a conjugated polymer in which double bonds and triple bonds are alternately present in the polymer main chain, and the diacetylene monomers are in crystalline or semi-crystalline form. When it is in close proximity to have a structure of having a feature made by irradiating ultraviolet or gamma rays.

Under optimal conditions, when polydiacetylene is dispersed in an aqueous solution or present in a thin film on a solid substrate, it exhibits a maximum absorption wavelength at about 650 nm. The blue polydiacetylene thus prepared is changed into a red solution having a maximum absorption wavelength at about 550 nm by the appropriate external environment change. For example, heating a blue polydiacetylene solution converts the color of the solution from blue to purple to red (Kim & Ahn et. Al., Macromolecules, 38: 9366, 2005). This color transition phenomenon from blue to red is not only the temperature but also the molecular recognition (USP 6,001,556; USP 6,180,135; USP 6,080,423) and the pH change (Ahn & Kim et. Al. Journal of the American Chemical Society, 125: 8976, 2003). It is also known to be caused by a variety of sensors, such as a variety of sensors have been reported using such a metabolic phenomenon, and in recent years, blue polydiacetylene does not fluoresce, but also a sensor using a feature that becomes fluorescence when the color transition occurs in red (Kim & Ahn et. Al., Journal of the American Chemical S ° C.iety, 127: 17580, 2005).

The present inventors have been continuously studying a temperature sensor capable of measuring an accurate temperature inside a microchannel of a microfluidic chip, and when using polydiacetylene having the above-described characteristics as a temperature sensor in a microfluidic chip, The temperature can be measured, and polydiacetylene is easily manufactured in aqueous solution, so it can be easily applied to the study of water-soluble biomaterials. Unlike other temperature sensors requiring complicated manufacturing process, the temperature can be measured only by fluorescence intensity observation. Knowing the merit of being able to measure, it came to complete this invention.

An object of the present invention is to use a temperature-sensitive fluorescent conjugated polymer as a temperature sensor in a microfluidic chip, so that the temperature can be measured in a very small amount, and is easily applicable to the study of water-soluble biomaterials, and other temperatures requiring complex manufacturing processes. Unlike sensors, microfluidic chips containing a temperature-sensitive fluorescent conjugated polymer as a temperature sensor and a microfluidic fluid using a temperature-sensitive fluorescent conjugated polymer as a temperature sensor, which can accurately measure the temperature in the microchannel of the microfluidic chip only by fluorescence intensity observation. The present invention provides a method for measuring the temperature of microchannels in a chip.

In order to achieve the above object, the present invention provides a method for measuring the temperature of the microchannel in the microfluidic chip using a temperature-sensitive fluorescent conjugated polymer as a temperature sensor.

As the temperature-sensitive fluorescent conjugated polymer that can be used in the present invention, a conjugated polymer that exhibits fluorescence in response to temperature such as polydiacetylene, polythiophene, or the like can be used.

Hereinafter, a method of measuring a temperature of a microchannel in a microfluidic chip using a temperature-sensitive fluorescent conjugated polymer according to the present invention as a temperature sensor will be described in detail.

The temperature measurement method of the microchannel in the microfluidic chip using the temperature-sensitive fluorescent conjugated polymer according to the present invention is to use the temperature-sensitive fluorescent conjugated polymer as the temperature sensor for accurate temperature measurement in the microchannel of the microfluidic chip. It features.

As the temperature-sensitive fluorescent conjugated polymer that can be used in the present invention, polydiacetylene, polythiophene, or the like can be used.

In one embodiment of the present invention, the method for measuring the temperature of a microchannel in a microfluidic chip according to the present invention comprises injecting a temperature-sensitive fluorescent conjugated polymer into the microchannel of the microfluidic chip and then generating the temperature by the temperature of the microchannel. The temperature of the microchannel can be measured by measuring the fluorescence intensity of the sensitized fluorescent conjugated polymer. This is because the temperature-sensitive fluorescent conjugated polymer fluoresces when exposed to heat.

In order to use the temperature-sensitive fluorescent conjugated polymer as a temperature sensor, a temperature-sensitive fluorescent conjugated polymer solution is injected into a microchannel of a microfluidic chip in the form of droplets, or a temperature-sensitive fluorescent conjugated polymer is formed on a microfluidic chip substrate. It can be used as a temperature sensor by depositing, it is preferable to inject the temperature-sensitive fluorescent conjugated polymer in the form of sensor droplets in the microchannel of the microfluidic chip.

In order to inject the temperature-sensitive fluorescent conjugated polymer into a micro-channel in the form of sensor droplets, sensor droplets are added at the intersections of the inlet channels by injecting a temperature-sensitive fluorescent conjugated polymer solution and oil into separate inlet channels to each inlet channel of the microfluidic chip. And when it is injected into the microchannel of the microfluidic chip with a syringe pump, the sensor droplet fluoresces by the temperature in the microchannel.

As shown in Test Examples 1, 3 and 4 below, in the method for measuring the temperature of the microchannel in the microfluidic chip according to the present invention, the polydiacetylene sensor droplets are constant as the temperature in the microchannel of the microfluidic chip increases. It can be seen that the fluorescence intensity increases. Therefore, the temperature in the microchannel can be measured by measuring the fluorescence intensity of the polydiacetylene sensor droplets.

As such, when a temperature-sensitive fluorescent conjugated polymer such as polydiacetylene is used as a temperature sensor in a microfluidic chip, the temperature in the microchannel of the microfluidic chip can be accurately measured simply by fluorescence intensity measurement.

The present invention also provides a microfluidic chip comprising a temperature sensitive fluorescent conjugated polymer as a temperature sensor.

Temperature-sensitive fluorescent conjugated polymers that can be used in the present invention include, but are not limited to, polydiacetylene, polythiophene, and the like.

In the microfluidic chip according to the present invention, various methods of integrating a temperature sensitive fluorescent conjugated polymer into a microfluidic chip as a temperature sensor may be used, but after the temperature sensitive fluorescent conjugated polymer is prepared from an aqueous solution of temperature sensitive fluorescent conjugated polymer, It is preferable to inject in the form of droplets into the microchannel and use it as a temperature sensor.

In one embodiment of the present invention, after preparing a PDMS microfluidic chip having three inlet channels and one microchannel by using a polydimethylsiloxane (PDMS) molding technology, the temperature sensitive to the center channel of the three inlet channels Injecting aqueous solution of fluorescent conjugated polymer and injecting oil into the remaining two channels to form sensor droplets at the intersections of the three inlet channels and introducing them into the micro-channel, thereby producing a microfluidic chip in which the temperature-sensitive fluorescent conjugated polymer is applied as a temperature sensor. Can be.

Edible oil, silicone oil, olive oil, etc. may be used as the oil, and there is no particular limitation as long as it is a solvent that is not mixed with water.

In another embodiment of the present invention, the microfluidic chip according to the present invention may include the temperature-sensitive fluorescent conjugated polymer in the form of a stream or a microstructure in the microfluidic chip. Further, the temperature-sensitive fluorescent conjugated polymer may be manufactured as a thin film or a fiber using a deposition method on a microfluidic chip and used as a temperature sensor.

In a microfluidic chip comprising the temperature-sensitive fluorescent conjugated polymer of the present invention as a temperature sensor, the temperature-sensitive fluorescent conjugated polymer is used as a temperature sensor for measuring temperature after being injected into the microchannel of the microfluidic chip in the form of droplets.

The sensor droplet formed of the temperature-sensitive fluorescent conjugated polymer has a characteristic that the fluorescence intensity increases linearly with temperature as described above, so that the sensor is flowed in the center of the microchannel of the microfluidic chip as in the present invention. When measuring the fluorescence intensity of the droplets can be accurately measured the temperature of the microfluidic chip micro-channel by an optical measuring method.

The present invention also provides a method of using a temperature-sensitive fluorescent conjugated polymer as a temperature sensor for measuring the temperature of the microchannel of the microfluidic chip.

As the temperature-sensitive fluorescent conjugated polymer that can be used in the present invention, polydiacetylene, poly thiophene, or the like can be used.

In one embodiment of the present invention, the polydiacetylene is a step of preparing a diacetylene monomer mixed solution by dissolving the diacetylene monomer of Formula 1 in water or a mixed solution of an organic solvent and water (step 1); Dispersing the diacetylene monomer by performing ultrasonic treatment on the mixed solution of diacetylene monomer prepared in step 1 (step 2); And a step (step 3) of polymerizing the diacetylene monomer by performing exposure treatment using ultraviolet rays to the mixed solution in which the diacetylene monomer is dispersed in the step 2;

[Formula 1]

In Chemical Formula 1,

d + g is 0,1 or 2, e + f is an integer from 2 to 50, e and f are integers greater than 1,

A and B are methyl group, amine group, carboxyl group, hydroxy group, maleimide group, biotin group, N-hydroxysuccinimide group, benzoic acid group or activated ester group,

L ₁ and L ₂ are the same as or different from each other and are an alkyl group having 2 or more carbon atoms, at least one ethylene oxide group, an amine group, an amide group, an ester group or a carbonyl group.

As the organic solvent, dimethylformamide (DMF), dimethylsuloxide (DMSO), chloroform, dichloromethane, hexane, tetrahydrofuran (THF), acetone, alcohol, mixtures thereof, and the like may be used. However, the present invention is not limited thereto.

The exposure treatment using ultraviolet rays in step 2 is preferably carried out by irradiating 220 to 300 nm ultraviolet rays for 1 to 10 minutes with respect to the mixed solution in which the diacetylene monomers are dispersed in the polymerization reaction of the diacetylene monomers.

The present invention is capable of measuring the temperature in a very small amount, and has a characteristic that the temperature-sensitive fluorescent conjugated polymer is easily prepared in an aqueous solution, so that it can be easily applied to the study of water-soluble biomaterials, and other temperature sensors requiring a complicated manufacturing process. Provides a microfluidic chip temperature measurement method comprising a microfluidic chip comprising a temperature-sensitive fluorescent conjugated polymer as a temperature sensor and a microfluidic chip using a temperature-sensitive fluorescent conjugated polymer as a temperature sensor. do.

Hereinafter, preferred examples are provided to help the understanding of the present invention, but the following examples are merely for exemplifying the present invention, and various changes and modifications within the scope and spirit of the present invention are apparent to those skilled in the art. It is natural that such variations and modifications fall within the scope of the appended claims.

< Example >

Example  One: Of microfluidic chips  Produce

As a microfluidic chip, a PDMS substrate having a microchannel pattern was prepared. Then, polydiacetylene was dissolved in water to prepare an aqueous polydiacetylene solution and injected into the center inlet channel of the microfluidic chip, and edible oil was injected into both side channels to generate sensor droplets at the cross point. The microfluidic chip was applied as a temperature sensor capable of sensing the temperature by flowing into the microchannel of the chip. At this time, the flow of the sensor droplet was accurately controlled using a syringe pump.

Test Example  One: Microfluidic Chip  My sensor Droplet Fluorescence intensity  Measure

(1) equipped with a micro heater and thermocouple Microfluidic Chip  Produce

In Test Example 1, a micro heater capable of controlling temperature and a film type thermocouple capable of measuring temperature are provided in order to measure fluorescence intensity according to an increase in temperature of a polydiacetylene sensor droplet in a microfluidic chip according to the present invention. The microfluidic chip was prepared as follows.

1 is a view schematically showing the structure of a microfluidic chip designed to measure the fluorescence intensity of polydiacetylene sensor droplets with increasing temperature inside the microchannel of the microfluidic chip in Test Example 1, wherein the microfluidic chip has a temperature It includes a micro heater that can control the and a film type thermocouple capable of measuring the temperature.

First, the microfluidic chip 100 was fabricated by bonding a glass wafer in which a PDMS substrate having a microchannel pattern, an ITO film micro heater 140, and a thermocouple 150 in a film form were integrated. PDMS substrates were fabricated using well-known soft lithography and molding techniques. Briefly, a mold was prepared by patterning a negative photoresist SU-8 50 on a silicon wafer in the shape of a microfluidic channel. The PDMS mixed solution was poured onto the prepared mold, the internal air was removed under vacuum for 30 minutes, and then dried in an oven for 2 hours to complete the PDMS substrate.

The micro heater 140 and the thermocouple 150 were integrated on the glass wafer in the following manner. Clean the glass wafer in acetone, isopropyl alcohol, deionized water in order, and then pattern the positive PR AZ4620 on it by photolithography to obtain a metal wire of the desired shape. Using a sputter to deposit a semi-transparent metal ITO to 2000Å thickness, and remove the remaining photoresist (PR) by a lift-off process using acetone, a micro heater having a metal wire of 50 μm width (140) was completed.

The thermocouple 150 in the form of a film was manufactured on a glass wafer with copper and constantan. The photoresist was patterned into a thermocouple in the same manner as the micro heater 140 was manufactured, the 200 nm copper wire was patterned by an electron beam evaporator, and unnecessary photoresist was removed by a lift-off process. Constantan wire was also fabricated in the same way as copper wire, except that an evaporator was used instead of a thermal evaporator. In order to protect the completed thermocouple 150 and the micro heater 140, a 800 nm SiO ₂ film was deposited using a sputter to insulate them.

(2) Microfluidic Chip  of mine Polydiacetylene  sensor Droplets  produce

In order to measure the temperature of the microchannel 100, the polydiacetylene sensor droplet 110 may be integrated into the microfluidic chip in various ways. However, in Test Example 1, a sensor was used in the form of droplets. The polydiacetylene aqueous solution was injected into the inlet channel among the three inlet channels 120 in the microfluidic chip 100 manufactured as described above, and edible oil was injected into both side channels to generate the sensor droplet 110. They were controlled by a syringe pump and introduced into the micro channel 130. 1 and 2, when two non-mixing fluids meet at an intersection, sensor droplets are generated due to flow instability.

(3) Of microfluidic chips  Temperature change inside the microchannel and thus the sensor Droplet  Fluorescence intensity measurement

The micro heater 140 integrated on the channel bottom surface while flowing the sensor droplet 110 generated as described above using a syringe pump from the inlet to the outlet in the microchannel 130 of the microfluidic chip 100. Was heated using electricity to linearly change the temperature in microchannel 130 from inlet to outlet. At this time, by measuring the temperature with the film-type thermocouple 150 produced at the inlet and outlet of the microchannel 130, the temperature of the middle of the microchannel 130 could be accurately known.

More specifically, the temperature in the microchannel 130 was measured using the thermocouple 150 while varying the voltage supplied to the micro heater 140 using the power supply 170. At the same time, the fluorescence intensity generated from the sensor droplet 110 in the microchannel 130 of the microfluidic chip 100 was recorded by using a fluorescence microscope and a CCD camera attached thereto. This is shown in Figure 4 by graphing. As shown in FIG. 3, the fluorescence intensity was changed according to the temperature in the microchannel 130 of the microfluidic chip 100. Referring to FIG. 4, when the temperature inside the microchannel 130 of the microfluidic chip 100 is changed from 40 ° C. to 60 ° C., it can be seen that the fluorescence intensity generated from the sensor droplet 110 increases constantly. It can be seen that the fluorescence intensity decreases when the temperature inside the microchannel 130 of the microfluidic chip 100 exceeds 70 ° C. Such measurement results can be used in various temperature ranges by using polydiacetylene synthesized using a diacetylene monomer of various structures as a temperature sensor.

1 is a view schematically showing the structure of a device designed to measure the fluorescence intensity of polydiacetylene sensor droplets with increasing temperature inside the microchannel of the microfluidic chip in Test Example 1.

Figure 2 is a photograph of observation of the formation of sensor droplets of polydiacetylene in the microfluidic chip using flow instability with an optical microscope.

3 is a photograph of a fluorescence intensity change of sensor droplets according to temperature in the microfluidic chip microchannel in Test Example 1 using a color CCD camera. In FIG. 3, the numbers on the right represent the amount of heat applied to the micro heater and the channel temperature at the observed point.

Figure 4 is a graph showing the fluorescence intensity change of the sensor droplets with temperature in the microfluidic chip microchannel in Test Example 1.

Explanation of symbols on the main parts of the drawings

100: device for measuring the fluorescence intensity of polydiacetylene sensor droplets with increasing temperature in the microchannel of the microfluidic chip

110: sensor droplet 120: injection hole

130: micro channel 140: micro heater

150: thermocouple 160: temperature measuring instrument

170: voltage supply

Claims (14)

Temperature measurement method of microchannel in microfluidic chip using temperature sensitive fluorescent conjugated polymer as a temperature sensor. The method according to claim 1, The temperature-sensitive fluorescent conjugated polymer is a polydiacetylene, characterized in that the microchannel in the microfluidic chip temperature measuring method. The method according to claim 1, Characterized in that the microchannel temperature is measured by injecting the temperature-sensitive fluorescent conjugated polymer into the microchannel of the microfluidic chip and then measuring the fluorescence intensity of the temperature-sensitive fluorescent conjugated polymer generated by the temperature of the microchannel. Temperature measurement method of micro channel in a floating chip. The method according to claim 1, In the method of injecting the temperature-sensitive fluorescent conjugated polymer into the microchannel of the microfluidic chip, a temperature-sensitive fluorescent conjugated polymer solution and an oil are added to each of the inlet channels of the microfluidic chip to form a sensor droplet at the inlet of the microchannel and then injected. A method of measuring the temperature of a microchannel in a microfluidic chip, characterized in that it is carried out. The method according to claim 1, The temperature-sensitive fluorescent conjugated polymer is characterized in that the fluorescence intensity increases constantly as the temperature increases in the microchannel of the microfluidic chip, temperature measurement method of the microchannel in the microfluidic chip. A microfluidic chip comprising a temperature sensitive fluorescent conjugated polymer as a temperature sensor. The method according to claim 6, The temperature-sensitive fluorescent conjugated polymer is a microfluidic chip, characterized in that polydiacetylene. The method according to claim 6, The temperature sensor is formed by injecting a temperature-sensitive fluorescent conjugated polymer solution in the form of droplets into the microchannel of the microfluidic chip or by depositing a temperature-sensitive fluorescent conjugated polymer in the form of a thin film on the microfluidic chip substrate. Microfluidic chip, characterized in that. The method according to claim 6, The temperature sensor is a microfluidic chip, characterized in that for measuring the temperature by measuring the fluorescence intensity generated by the temperature in the microchannel of the microfluidic chip. A method of using a temperature sensitive fluorescent conjugated polymer as a temperature sensor for measuring the temperature of a microchannel of a microfluidic chip. The method according to claim 10, The temperature-sensitive fluorescent conjugated polymer is polydiacetylene, characterized in that the method of using a temperature-sensitive fluorescent conjugated polymer as a temperature sensor for measuring the temperature of the microchannel of the microfluidic chip. The method of claim 11, The polydiacetylene is prepared by dissolving a diacetylene monomer represented by Chemical Formula 1 in water or a mixed solution of an organic solvent and water (step 1); Dispersing the diacetylene monomer by performing ultrasonic treatment on the mixed solution of diacetylene monomer prepared in step 1 (step 2); And a step (3) of polymerizing the diacetylene monomer by performing exposure treatment using ultraviolet rays to the mixed solution in which the diacetylene monomer is dispersed in step 2, wherein the temperature-sensitive fluorescent conjugated polymer is fine. Use as a temperature sensor to measure the temperature of the microchannels in the flow chip: [Formula 1]

In Chemical Formula 1, d + g is 0,1 or 2, e + f is an integer from 2 to 50, e and f are integers greater than 1, A and B are methyl group, amine group, carboxyl group, hydroxy group, maleimide group, biotin group, N-hydroxysuccinimide group, benzoic acid group or activated ester group, L ₁ and L ₂ are the same as or different from each other and are an alkyl group having 2 or more carbon atoms, at least one ethylene oxide group, an amine group, an amide group, an ester group or a carbonyl group. The method according to claim 12, The organic solvent is selected from the group consisting of dimethylformamide, dimethyl sulfoxide, chloroform, dichloromethane, hexane, tetrahydrofuran, acetone, alcohol and mixtures thereof, temperature-sensitive fluorescent conjugated polymer microfluidic chip A method for use as a temperature sensor for measuring the temperature of a microchannel. The method according to claim 12, The exposure treatment using ultraviolet rays in step 2 is carried out by irradiating 220 to 300 nm ultraviolet rays for 1 to 10 minutes to the mixed solution in which the diacetylene monomer is dispersed, the temperature-sensitive fluorescent conjugated polymer microfluidic chip A method for use as a temperature sensor for measuring the temperature of a microchannel.

Images