Patent Description:
Efficient amplification of target nucleic acid is a very important factor for detection of nucleic acid as well as DNA sequencing, cloning, etc. There are various methods for amplifying nucleic acid such as PCR(polymerase chain reaction), LCR(ligase chain reaction), SSR(self-sustained sequence replication), NASBA(nucleic acid sequence based amplification), and SDA(strand displacement amplification), etc..

Many of the above methods have disadvantages in that they have a relatively low accuracy of quantitative measurement and requires expensive devices and the disadvantages are particularly getting worse when it is needed to analyze more than one target simultaneously.

To make up for such disadvantages, an isothermal amplification was invented and in particular, a RCA (rolling circle amplification) method has been paid much attention to. That is, several techniques such as PCR have been used for detecting DNA so far, but these methods are disadvantageous since they are time-consuming, are not efficient and require high cost and manpower.

PCR method comprises a denaturation in which DNA is separated into single strand by heating DNA in reaction solution containing primer pairs, template, polymerase, and dNTP; an annealing in which a primer complementary to each separated DNA chain is bonded to a template by lowering temperature; and a polymerization in which new strands is polymerized by a polymerization reaction using polymerase by increasing temperature, and such an amplification increases DNA chain exponentially.

However, PCR process should have experience the above steps and thus, it inevitably incurs temperature change. Therefore, devices for PCR must have a temperature controller and a heating means. If PCR is used for the amplification of target nucleic acid in a lab-on-a-chip (LOC), etc., a temperature controller and a heater for PCR reaction is further required besides detection devices for LOC, it is disadvantageous that devices are complex and cost for the devices is increasing.

To solve the above disadvantage, several isothermal amplification methods have been suggested. LAMP (loop-mediated isothermal amplification) is one of the isothermal amplification methods and generates a multi-loop product having branches using <NUM> amplification primers. Such LAMP is not appropriate for an early diagnosis or a biosensor since an initial reverse transcriptase (RT) is used to detect a target RNA.

RCA method was suggested as another isothermal amplification method. Advantageously, RCA method does not require a temperature change necessary for the PCR amplification as described above and thus, it is possible to amplify a target nucleic acid in a isothermal state. Therefore, it is possible to amplify without a temperature controller in a process requiring the amplification, thereby reducing a complexity of the device and costs.

In a LRCA (linear rolling circle amplification) method, a target NDA sequence and an open circular probe are hybridized to form a complex and then an amplification target circle is generated by ligation, and thereafter, primer sequence and DNA polymerase are introduced. Amplification target circle forms a template in which a new DNA is formed, elongation is carried out from primer to be extended into continuous sequence of repeated sequence complementary to an amplification target circle, thereby generating thousands of copies of nucleic acid per an hour.

As a further developed method, ERCA(exponential RCA) method was developed. In the ERCA method, a new amplification center is provided using an additional primer sequence which bonds to a replicated sequence complementary to an amplification target circle, thereby the amplification is increased exponentially. In the ERCA method, a strand displacement method is continued, but it is limited to a method in which an initial single strand RCA product is used as a template of another DNA synthesis by using a separate single strand primer attached to the product without additional RCA.

Another method using molecular padlock probe(MPP) and rolling circle amplification (RCA) is disclosed (<NPL>). This method has several advantages and is characterized in that complementary nucleic acid is amplified in a circular MPP by the identification of target nucleic acid sequence and a high specificity. In particular, sensitivity is improved by direct coupling of RCA product without further purification. RCA reaction can be started on the surface by fixing target nucleic acid probes to a surface of material such as gold, quartz, etc. through simple chemical surface treatment.

Meanwhile, paper published by <NPL>) discloses a technique in which RCA reaction surface is provided on the bottom of the microchannel, then for two hours, a reaction with sample solution is carried out, and a single strand of DNA is elongated and self-assembly in the form of multiple dumbbells occurs, thereby DNA becomes to form a hydrogel shape and a flow in the corresponding channel is excluded.

However, techniques disclosed in the paper of Ho Yeon Lee have a disadvantage that it takes more than two hours to take a test, since RCA reaction surface should be formed on the bottom surface of the microchannel and then a tester has to wait until the entire microchannel is blocked by the amplification at the RCA reaction surface.

An example of a microfluidic device for detecting a target gene is known from publication <NPL> as well as from <CIT> and <CIT>.

<CIT> discloses an agglutination based assay system for determining the presence and/or amount of analyte in a sample comprising a test device having one or more capillary pathways comprising detection regions adapted for non-visual detection of a sample which is releasably engageable with a reader which comprises detection means for detecting the sample at the detection regions in each of said capillary pathways and electronic means for indicating the presence and/or amount of analyte.

<NPL> discloses binding partners to detect genes by changes in flow through capillary.

<NPL>provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions.

Accordingly, the present invention is provided to solve the above problems and an object of the present invention is to provide a microfluidic device for detection target gene which can reduce the detection time significantly, wherein for the detection of various pathogenic viruses by the detection of genes, the target gene is detected by phenomenon in which voids generated by the microbeads are blocked or a size of the voids is reduced by the amplification of the target gene.

Another object of the present invention is to provide a microfluidic device for detecting the target gene, wherein it is possible to quantitatively analyze the target gene according to the detection based on various methods.

The above objects are solved by the claimed matter according to the independent claim.

According to the present invention, a microfluidic device for detecting a target gene, comprises: a sample chamber containing sample solution; a microchannel which is connected to the sample chamber and through which the sample solution flows; and a microbead packing arranged on a flow path of the sample solution in the microchannel; wherein the microbead packing comprises: a packing tube arranged at a microchannel so as to partially constitute the flow path of the sample solution, a plurality of microbeads contained in the packing tube and being in close contact with each other to form voids between the microbeads, and probe linkers formed on a surface of each microbead, wherein the probe linkers are configured to amplify a target gene in the sample solution by complementary bonding with the target gene, thereby detecting the target gene, according to another embodiment of the present invention.

Here, the void may be blocked or a size of the void may be reduced by the amplification of the target gene induced by the complementary bonding between the target gene and the probe linker, whereby a final travel distance of the sample solution, time taken for the final travel distance, and a flow rate of the sample solution are changed, and wherein the target gene may be detected by one of the final travel distance, the time taken for the final travel distance and the flow rate.

According to the invention, the microfluidic device further comprises a negative pressure chamber arranged on the opposite side of the sample chamber to be in fluid communication with the microchannel, the negative chamber applying negative pressure from outside to make the sample solution flow through the microchannel, wherein the void is blocked or a size of the void is reduced by the amplification of the target gene induced by the complementary bonding between the target gene and the probe linker, whereby the target gene is detected according to a change of the negative pressure applied by the negative pressure chamber.

Also, a diameter of the microbead may be set to have a size such that the target gene is able to pass through the void according to a type of the target gene within the range of <NUM> to <NUM>.

Also, the microbead packing may comprise meshes arranged respectively at both ends of the packing tube to prevent loss of the microbeads.

According to the invention, the sample chamber, the microchannel and the microbead packing are provided in plural, respectively to be arranged in parallel and the microbeads contained in one of the microbead packings do not have the probe linkers.

Also, the sample chamber, the microchannel and the microbead packing may be provided in plural, respectively to be arranged in parallel and the probe linkers of each microbead packing are configured to detect different target genes.

Also, the microchannel may comprise: a first flow channel connected to the sample chamber and a plurality of second flow channels diverged from the first flow channel, wherein the microbead packing may be provided in plural to be arranged in each of the second flow channels, wherein the microbeads contained in each microbead packing may have a different diameter.

Also, the probe linker may comprise a coating part coated on a surface of the microbead, a primer attached to the coating part, and a template which complementarily bonds to the primer, wherein the template may comprise a first bonding part which complementarily bonds to the target gene, a second bonding part which complementarily bonds to the primer, and a third bonding part which is complementary in the template to form a dumbbell, and wherein the first bonding part is divided and formed at both ends of the template and the second bonding part is formed between the third bonding part which are divided.

Also, the coating part may include one or more selected from a group consisting of <NUM>-hydroxydopamine hydrochloric acid, norepinephrine, epinephrine, pyrogallolamine, DOPA(<NUM>,<NUM>-Dihydroxyphenylalanine), catechin, tannins, pyrogallol, pyrocatechol, heparin-catechol, chitosan-catechol, poly(ethylene glycol)-catechol, poyl(ethyleneimine)-catechol, poly(methylmethacrylate)-catechol, hyaluronic acid-catechol, polylysine-catechol, and polylysine.

Also, the primer may include one or more selected from a group consisting of thiol, amine, hydroxyl, carboxyl, isothiocyanate, NHS ester, aldehyde, epoxide, Carbonate, HOBtester, Glutaraldehyde, carbamate, imidazole carbamate, maleimide, aziridine, sulfone, vinylsulfone, hydrazine, phenyl azide, benzophenone, anthraquinone, and Diene groups, and wherein a terminal of the primer is modified.

By the above arrangement, for the detection of various pathogenic viruses by the detection of genes, the target gene is detected by phenomenon in which voids generated by the microbeads are blocked or a size of the voids is reduced by the amplification of the target gene, thereby making it possible to provide a microfluidic device for detection target gene which can reduce the detection time significantly.

Also, it is possible to quantitatively analyze the target gene according to the detection based on various methods.

The present invention relates to a microfluidic device for detecting a target gene and is characterized in that it comprises: a plurality of capillary tubes which are partially immersed in a sample container containing sample solution and in which the sample solution flows by capillary phenomenon, and microbead packings arranged at one part in each capillary tube to be arranged on a flow path of the sample solution, wherein each of the microbead packings comprises: a packing tube arranged at the capillary tube so as to partially constitute the flow path of the sample solution, a plurality of microbeads contained in the packing tube and being in close contact with each other to form voids between the microbeads, and probe linkers formed on a surface of each microbead, wherein the probe linkers are configured to amplify a target gene in the sample solution by complementary bonding with the target gene, thereby detecting the target gene, according to the present invention.

<FIG> is a view illustrating a microfluidic device <NUM> for detecting target gene according to an embodiment outside the scope of the claimed invention.

Referring to <FIG>, the microfluidic device <NUM> for detecting target genes according to an embodiment outside the scope of the claimed invention comprises a plurality of capillary tubes <NUM> and microbead packings <NUM>.

The plurality of capillary tubes <NUM> are configured to have a size such that sample solution <NUM> is able to flow into the capillary tube by capillarity phenomenon. In the embodiment, for example, as shown in <FIG>, six capillary tubes are arranged to be adjacent to each other, but the arrangement of the tubes is not limited thereto.

The plurality of capillary tubes <NUM> are configured such that one end portion of the tube, i.e., a lower part of the tube as shown in <FIG>, is immersed in a sample container <NUM> which contains the sample solution <NUM>, and the sample solution <NUM> flows into the tube through an inlet at the lower side of the tube by capillarity phenomenon and flows upwards.

Microbead packing <NUM> is arranged in each capillary tube <NUM> on a flow path of the sample solution <NUM>. Here, for example, the microbead packing <NUM> is manufactured separately from the microfluidic device <NUM> and then is inserted into the capillary tube <NUM> when the microfluidic device is manufactured.

Meanwhile, the microbead packing <NUM> according to the embodiment may comprise a packing tube <NUM>, a plurality of microbeads <NUM> and probe linkers <NUM>, as shown in <FIG>.

The packing tube <NUM> is arranged on a flow path of the sample solution <NUM>, i.e., in the capillary tube <NUM>, to constitute some of the flow path of the sample solution <NUM>. Also, the plurality of microbeads <NUM> contained in the packing tube <NUM> are in close and tight contact with each other and there are voids between the microbeads which are in close and tight contact with each other.

Here, a diameter of the microbead <NUM> depends on the type of the target gene and the size of the void varies according to a diameter of the target gene. In this regard, the size of the void is set to make the target gene pass through the void and the size of the void can be adjusted by adjusting the size of the microbeads <NUM>. In the embodiment, for example, the diameter of the microbead <NUM> is determined according to the type of the target gene within the range of <NUM>. 1µmto <NUM>.

Further, the microbead packing <NUM> according to the embodiment may comprise mesh <NUM> (see the embodiment in <FIG>) which are arranged respectively at both ends of the packing tube <NUM>. Advantageously, the inner diameter of the mesh <NUM> has a size by which the mircobead <NUM> is not discharged and the flow of the sample solution <NUM> is not disturbed. Meanwhile, the probe linker <NUM> is formed on a surface of each microbead <NUM>. Also, the probe linker <NUM> is configured such that amplification by the complementary bond with the target gene in the sample solution <NUM> is carried out, thereby detecting the target gene.

In the embodiment, the probe linker <NUM> comprises constituents disclosed in a paper '<NPL>, et al. That is, for example, the probe linker <NUM> comprises a coating part, a primer, and a template.

The coating part is coated on the surface of the microbead <NUM> and is formed of material to which the primer is attached and fixed. As described in the above paper, examples of the coating part may include one or more selected from a group of <NUM>-hydroxydopamine hydrochloric acid, norepinephrine, epinephrine, pyrogallolamine, DOPA(<NUM>,<NUM>-Dihydroxyphenylalanine), catechin, tannins, pyrogallol, pyrocatechol, heparin-catechol, chitosan-catechol, poly(ethylene glycol)-catechol, poyl(ethyleneimine)-catechol, poly(methylmethacrylate)-catechol, hyaluronic acid-catechol, polylysine-catechol, and polylysine, etc..

The primer is fixed at the coating part and the template is complementarily bonded to the primer. Here, the template may comprises a first bonding part which is complementarily bonded to the target gene, a second bonding part which is complementarily bonded to the primer, and a third bonding part which is complementary in the template to form a shape of dumbbell. Further, the first bonding part is divided and formed at both ends of the template and the second bonding part is formed between the third bonding parts which are divided.

Here, examples of the primer may include one or more selected from a group consisting of thiol, amine, hydroxyl, carboxyl, isothiocyanate, NHS ester, aldehyde, epoxide, Carbonate, HOBtester, Glutaraldehyde, carbamate, imidazole carbamate, maleimide, aziridine, sulfone, vinylsulfone, hydrazine, phenyl azide, benzophenone, anthraquinone, and Diene groups, wherein a terminal is modified.

With the above arrangement, the target gene is bonded to the probe linker <NUM> of the embodiment and is amplified and the amplified target gene is to form hydrogel. Detailed explanation will be omitted since it is disclosed in the above paper.

As described above, an amplification by a complementary bonding of a target gene to a probe linker <NUM> formed on the surface of each microbead <NUM> generates a hydrogel, thereby blocking the void between the microbeads <NUM> and reducing the size of the void. The blocking of the void and the reduction of void size will be a resistance and in turn will prevent arise of the sample solution <NUM> due to capillary phenomenon or reduce the speed of the rise, thereby detecting a target gene.

Referring to <FIG>, a method of detecting a target gene using the microfluidic device <NUM> according to the above embodiment will be described.

First, as shown in <FIG>, the sample solution <NUM> is poured into the sample container <NUM>. Here, an initial quantity (WL1) of the sample solution <NUM> injected into the sample container <NUM> will be set to make the sample solution <NUM> reach the microbead packing <NUM> through the capillary tube <NUM> by capillary phenomenon (see CL1).

Here, the target gene in the sample solution <NUM> passing through the microbead packing <NUM> is amplified by the complementary bonding with the probe linker <NUM> of the microbead packing <NUM> and in turn, the target gene becomes hydrogel, thereby blocking the void between the microbeads <NUM> and reducing the size of the void.

Here, in case that probe linkers <NUM> of each microbead packing <NUM> are provided to detect different target genes, only the void of the microbead packing <NUM> having the corresponding target gene will be blocked or a size of the void of the microbead packing <NUM> having the corresponding target gene will be reduced.

Then, as shown in <FIG>, when the sample solution <NUM> is further supplied into the sample container <NUM> (see WL2), the sample solution <NUM> is supposed to flow upwards through the capillary tube <NUM>. Here, if the sample solution <NUM> cannot flow to reach the opposite side of the microbead packing <NUM> by the amplification of the target gene, the presence of the corresponding target gene can be identified.

Referring to <FIG>, in the two capillary tube <NUM> on the left, it can be seen that the sample solution <NUM> passed through the microbead packing <NUM> and rose (see CL2a) and this shows that the sample solution <NUM> has no target gene which bonds with the probe linker <NUM> of the microbead packing <NUM>.

On the contrary, in the two capillary tube <NUM> on the right, it can be seen that the sample solution <NUM> did not pass through the microbead packing <NUM> since the microbead packing <NUM> is blocked and this shows that the sample solution <NUM> has target gene which bonds with the probe linker <NUM> of the microbead packing <NUM>. And, in the capillary tube on the center, it can be seen that the sample solution <NUM> has relatively small quantity of target gene since the void was not blocked completely or it takes time for the void to be blocked.

In the above embodiment, it was tested whether the void is blocked by adding sample solution. In an alternative method, the capillary tube <NUM> is immersed in the sample solution, and then, the capillary tube <NUM> is immersed deeper in the sample solution after a period of certain time, for example, time for which target gene can be amplified enough. By this, if the sample solution <NUM> flowing upwards through the capillary tube <NUM> cannot flow to reach the opposite side of the microbead packing <NUM> due to the amplification of the target gene, it is possible to identify the presence of the target gene.

Here, in order to facilitate a visual identification as to whether the sample solution has moved to the upper part of the capillary tube <NUM>, the color of the sample solution can be controlled. For example, a paper whose color changes when it is wet is arranged at the upper part of the microbead packing <NUM> in the capillary tube <NUM>. By this arrangement, if color of the paper located in the capillary tube <NUM> is changed when the paper is wet by the sample solution, it can be identified that there is no target gene in the capillary tube <NUM>.

Hereinafter, referring to <FIG>, the microfluidic device <NUM> for detecting target gene according to another embodiment will be described in detail.

Referring to <FIG>, the microfluidic device <NUM> for detecting target gene according to another embodiment outside the scope of the claimed invention (hereinafter referred to as 'microfluidic device <NUM>') may comprise a microchannel <NUM> and a microbead packing <NUM>. Also, the microfluidic device <NUM> according to the embodiment may comprise a negative pressure chamber <NUM>.

The sample chamber <NUM> is arranged at one side of the microfluidic device <NUM> and contains sample solution. The microchannel <NUM> is in fluid communication with the sample chamber <NUM>, and the sample solution contained in the sample chamber <NUM> flows through the microchannel <NUM>.

The microbead packing <NUM> is arranged on a flow path of the sample solution. Here, the microbead packing is manufactured separately from the microfluidic device <NUM> and then is installed in the microchannel <NUM> when the microfluidic device <NUM> is manufactured. For example, when an upper substrate having a sample chamber <NUM>, a microchannel <NUM>, and a fluid-pressure chamber is attached to a transparent base substrate in an upward and downward direction in order to manufacture the microfluidic device <NUM>, the upper substrate is attached to the base substrate in a state that the microbead packing <NUM> was inserted into the microchannel <NUM> of the upper substrate.

Meanwhile, as shown in <FIG>, the microbead packing <NUM> according to the embodiment may comprise a packing tube <NUM>, a plurality of microbeads <NUM> and probe linkers <NUM>. Here, the microbead packing <NUM> corresponds to that of the embodiment in <FIG> and detailed explanation thereof is omitted. As shown in <FIG>, the microbead packing <NUM> according to the embodiment may comprise meshes <NUM> which are arranged on both ends of the packing tube <NUM> respectively to prevent the loss of the microbeads <NUM>. Advantageously, the size of the inner diameter of the mesh <NUM> is set to prevent the microbeads <NUM> from being discharged and not to disturb the flow of the sample solution.

Here, an amplification by a complementary bonding of a target gene to probe linkers133 formed on the surface of each microbead141 generates a hydrogel, thereby blocking the void <NUM> between the microbeads <NUM> and reducing the size of the void <NUM>, thereby causing changes of a final travel distance by which the sample solution flows through the microchannel <NUM>, time taken to reach the final travel distance, and a flow rate of the sample. Further, it is possible to detect the target gene by using at least one of the final travel distance, the travel time and the flow rate.

Referring back to <FIG>, the negative-pressure chamber <NUM> is arranged on the opposite side of the sample chamber <NUM> and is in fluid communication with the microchannel <NUM>. Negative pressure from outside is applied by the negative-pressure chamber <NUM> such that sample solution flows through the microchannel <NUM>.

Here, if the void <NUM> is blocked or the size of the void <NUM> is reduced by the amplification due to the complementary bonding of the target gene to the probe linker <NUM>, pressure which has been being constantly applied by the negative-pressure chamber <NUM> will change, thereby it being possible to identify the presence of the target gene.

According to the above embodiment, a plurality of microbeads <NUM> constitute the microbead packing <NUM>, and probe linkers <NUM> formed on the microbeads <NUM> bond with the target gene. Then, the target gene is amplified and hydrogel is generated during the amplification and blocks the void <NUM> or reduces the size of the void <NUM>, thereby causing the changes of the final travel distance, the travel time, the flow rate and/or pressure, whose detection makes it possible to identify the presence of the target gene.

Also, it is possible to make a test by blocking or clogging the void <NUM> formed by the microbeads <NUM> instead of blocking the entire of the microchannel <NUM> as disclosed in the paper, thereby making it possible to reduce test time significantly. Further, it is possible to increase a reaction area by forming probe linkers <NUM> on the surface of each microbeads <NUM>, instead of forming a reaction area on the bottom surface only as disclosed in the above paper, thereby making it possible to reduce test time significantly.

It is possible to detect the presence of the target gene by clogging phenomenon of the void, and it is also possible to evaluate quantitatively the target gene by using the change of the final travel distance, the travel time, the flow rate or the pressure, etc. For example, if there are a lot of target genes, the probability of reaction increases and in turn, the void <NUM> is blocked more quickly and the final travel distance is reduced, which can be quantified based on a statistical method, thereby making it possible to evaluate quantitatively the target gene.

<FIG> shows a microfluidic device 100a according to the present invention. As shown in <FIG>, the microfluidic device 100a according to the embodiment may comprise a plurality of sample chambers <NUM>, a plurality of microchannels <NUM>, and a plurality of microbead packings <NUM>. Here, the sample chamber <NUM> and the microchannel <NUM> are connected to each other by one and one to form a flow path of the sample solution and a plurality of flow paths are arranged in parallel. In <FIG>, for example, two sample chambers <NUM> and two microchannels <NUM> are arranged in parallel to form two flow paths. The microbead packings <NUM> are arranged respectively in each microchannel <NUM>.

Here, the microbeads <NUM> contained in one of the microbead packings <NUM> do not have probe linkers <NUM>. Referring to <FIG>, as described above, for example, the microbeads <NUM> of one of microbead packings <NUM> have probe linkers <NUM> and the microbeads <NUM> of the other of the microbead packings <NUM> do not have probe linkers <NUM>.

In the example, the same sample solution is contained in each sample chamber <NUM> and then the sample solution flows. Here, if the microbead packing <NUM> has the probe linkers, the bonding and the amplification of the target gene as described above cause the microbead packing to be blocked or cause the size of the void <NUM> to be reduced, thereby the flow of the sample solution being restricted. On the contrary, if the microbead packing <NUM> has no probe linkers <NUM>, the sample solution flowing through the microbead packing <NUM> flows without restriction.

Further, in case that the microbead packing <NUM> has the probe linkers <NUM>, the final travel distance by which the sample solution which passed through the microbead packing <NUM> has traveled until the sample solution is stopped by the blocking of the microbead packing <NUM> is measured and the travel time thereof is measured, and then the final travel distance and time thereof are compared with those of the microbead packing <NUM> having no probe linkers <NUM>, thereby it is possible to quantify an initial concentration of the target gene.

Here, in the embodiment of <FIG>, for example, each flow path is provided with a negative pressure chamber <NUM>. Besides, of course, it should be noted that an end of each microchannel <NUM> is connected to one negative pressure chamber <NUM>, and negative pressure is applied for the flow of the sample solution by one negative pressure chamber <NUM>.

Further, in the embodiment of <FIG>, microbeads <NUM> of each microbead packing <NUM> may have different probe linkers <NUM>, respectively so as to bond with different target genes. That is, since each probe linker <NUM> bonds with different target genes to amplify target genes, it is possible to detect a plurality of target genes simultaneously, by using one microfluidic device <NUM>.

<FIG> shows a microfluidic device 100b according to another embodiment of the present invention. The embodiment as shown in <FIG> is a modification of the embodiment of <FIG> and for example, each sample chamber <NUM> is provided with a stirrer <NUM>.

Here, the stirrer <NUM> is configured to rotate by a rotation of a magnet <NUM> arranged outside the microfluidic device <NUM>. By the stirrer, the sample solution contained in the sample chamber <NUM> is stirred and in turn, target genes in the sample solution is distributed widely, thereby facilitating the bonding and amplification in the microbead packing <NUM>.

<FIG> shows a microfluidic device 100c according to another embodiment outside the scope of the claimed invention.

Referring to <FIG>, a microchannel <NUM> of the microfluidic device 100c according to yet may comprise a first flow channel <NUM> which is connected to a sample chamber <NUM> andthe first flow channel <NUM> branches off into a plurality of second flow channels 132a,132b,132c,132d,132e. Further, microbead packings140a,140b,140c,140d,140e are arranged in the second flow channel 132a,132b,132c,132d,132e, respectively.

Here, the microbeads <NUM> contained in each microbeadpackings140a,140b,140c,140d,140e may have different inner diameter. The probe linkers <NUM> formed in each microbead <NUM> may be configured to bond with the same target gene.

In the above arrangement, microbeadpackings140a,140b,140c,140d,140e having small diameter of the microbead <NUM> may be blocked even when the concentration of target gene in the sample solution is low. The blocking of the microbeadpackings140a,140b,140c,140d,140edepends on the concentration of the target gene in the sample solution and in turn, the blocking of the microbead packings occurs according to the order of the diameter size of the microbead <NUM>. Accordingly, it is possible to evaluate quantitatively the target gene in the sample solution, based on the diameter of the microbead <NUM> which generates the blocking lastly.

Here, in the embodiment of <FIG>, for example, a plurality of second flow channels 132a,132b,132c,132d,132e are connected to each negative pressure chamber 120a,120b,120c,120d,120e, respectively. Alternatively, it is noted that ends of second flow channels 132a,132b,132c,132d,132e are merged to be connected to one negative pressure chamber.

Although several embodiments of the present invention are illustrated and explained above, it is obvious that the embodiments can be easily devised by those skilled in the technical idea of the present invention within the scope of the present invention. The scope of the present invention is determined by attached claims.

Claim 1:
A microfluidic device (<NUM>,<NUM>,100a,100b,100c) for detecting a target gene, comprising:
a sample chamber (<NUM>) adapted to contain sample solution;
a microchannel (<NUM>) which is connected to the sample chamber through which the sample solution flows; and
a microbead packing (<NUM>,<NUM>,140a,140b,140c,140d,140e) arranged on a flow path of the sample solution in the microchannel (<NUM>);
wherein the microbead packing (<NUM>,<NUM>,140a,140b,140c,140d,140e) comprises:
a packing tube (<NUM>,<NUM>) arranged at a microchannel (<NUM>) so as to partially constitute the flow path of the sample solution,
a plurality of microbeads (<NUM>,<NUM>) contained in the packing tube (<NUM>,<NUM>) and being in close contact with each other to form voids between the microbeads (<NUM>,<NUM>), and
probe linkers formed on a surface of each microbead (<NUM>,<NUM>),
wherein the probe linkers are configured to amplify a target gene in the sample solution by complementary bonding with the target gene, thereby detecting the target gene, characterized by further comprising a negative pressure chamber arranged on the opposite side of the sample chamber (<NUM>) to be in fluid communication with the microchannel (<NUM>), the negative chamber being arranged to apply negative pressure from outside to make the sample solution flow through the microchannel (<NUM>),
which is arranged so that the void is blocked or a size of the void is reduced by the amplification of the target gene induced by the complementary bonding between the target gene and the probe linker, whereby the target gene is detected according to a change of the negative pressure applied by the negative pressure chamber,
wherein the sample chamber (<NUM>), the microchannel (<NUM>) and the microbead packing (<NUM>,<NUM>,140a,140b,140c,140d,140e) are provided in plural, respectively to be arranged in parallel and the microbeads contained in one of the microbead packings (<NUM>,<NUM>,140a,140b,140c,140d,140e) do not have the probe linkers.