Patent Application
20070267336
With reference to the drawings, a description will hereinafter be made about preferred embodiments for practicing the present invention. It is to be noted that the embodiments to be described hereinafter merely illustrate examples of some representative embodiments of the present invention and the scope of the present invention shall not be narrowly interpreted by the preferred embodiments.
Referring first to
Roughly speaking, the microflow system designated at numeral 1 in
The illustrated microflow path system 1 is provided, inside a part of the capillary 11 at a location proximal to the inlet portion 12, with a hybrid-forming section 111 of the construction that a number of beads 2 of microdiameters are packed. This hybrid-forming section 111 functions as a section (region) in which a desired interaction such as hybridization is allowed to proceed.
A bead bed length B of the beads 2 packed in the hybrid-forming section 111 may desirably be set at 4 mm or shorter, with 2 mm or shorter being more preferred. In general, the longer the bead bed length B, the higher the internal pressure of the microflow path. In the microflow path system according to an embodiment of the present invention, the bead bed length B of the hybrid-forming section 111 is hence dimensioned short to prevent a rise in the internal pressure of the microflow path.
A fluid-supply promoting section 112 is arranged on a downstream side of the hybrid-forming section 111 (in other words, on the side of the outlet portion 13). This fluid-supply promoting section 112 has a role to accelerate the flow rate of a supply of a sample solution, washing buffer solution or the like to be supplied toward the hybrid-forming section 111. No particular limitation is imposed on particles 3 packed in the fluid-supply promoting section 112, insofar as they have a function to promote a fluid supply. As a preferred example, however, perfusion chromatography particles can be packed.
These perfusion chromatography particles typically have large pores called “through pores” and also small pores called “diffusive pores”. Owing to this structure, molecules dissolved in a buffer solution are allowed to pass through the through pores, and are carried to all corners of the diffusive pores. As a consequence, a large area of contact can be established between the molecules and functional groups on the surfaces of the filler and the distances between the flow of the buffer and the functional groups become very small (not greater than 1 μm) irrespective of the particle size of the filler. The fluid can, therefore, be supplied at high flow rate and under low pressure.
In addition to the promotion of a fluid supply, the arrangement of the fluid-supply promoting section 112 is also expected to bring about advantageous effects to be described hereinafter. In general, various members such as nuts for the connection with external pumps or the like are often provided at capillary portions adjacent to the inlet portion 12 and outlet portion 13. Accordingly, it becomes difficult to detect or measure if the hybrid-forming section 111 is provided in these portions. The arrangement of the fluid-supply promoting section 112 on the downstream side of the hybrid-forming section 111, however, makes it possible to arrange the hybrid-forming section 111, which is a target of a detection or measurement, at or around a center of the flow path system 1, thereby bringing about an advantage that the detection or measurement can be facilitated.
Further, selection of perfusion chromatography particles of an adequate type makes it possible to have any surplus substances and hazardous substance (for example, radioactive substance) adsorbed and trapped to prevent their discharge to the outside. Accordingly, the selection of such particles as the particles 3 can also bring about a merit that the solution passed through the microflow path system 1 can be discarded as is.
When supplying a sample solution into the microflow path system 1 according to an embodiment of the present invention, the sample solution can be supplied as is. It is, however, more preferred to supply the sample solution into the microflow path system 1 subsequent to its filtration. This filtration is intended to remove suspended substances and the like. If suspended substances and the like in a sample solution are filtered out in advance, the microflow path 1 can be prevented from clogging.
A filter for use in the filtration may desirably have a pore size of 0.65 μm or smaller, with 0.1 μm or smaller being more preferred, for the removal of suspended substances and long-chain nucleic acids. To improve the precision of filtration, centrifugation can be conducted as desired before the filtration.
In the microflow path system 1 according to an embodiment of the present invention, depressurization can be performed on a downstream of the fluid-supply promoting section 112. By a fluid supply from an upstream, a pressure generally builds up on an upper side of the hybrid-forming section 111. By keeping the downstream side under a negative pressure, the flow of the fluid through the microflow path system 1 can be improved.
Describing one example of the manner of depressurization, the outlet side can be maintained under a negative pressure by connecting a syringe or the like to the outlet portion 13 and pulling the plunger of the syringe although such a syringe or the like is not shown in the drawing.
With reference to
The beads 21 are small microbeads made of a material such as polystyrene. The surfaces of these beads 21 are equipped with a structure suited for chemically binding nucleic acid molecules at one ends thereof.
These beads 21 are provided with molecules P of a probe nucleic acid (which will hereinafter be simply called “the probe nucleic acid P”) bound on the surfaces of the beads 21, for example, via avidin-biotin bonds or through a coupling reaction (for example, a diazo coupling reaction). In this state, the beads 21 are packed in the hybrid-forming section of the microflow path system 1. In the hybrid-forming section 11.1, the probe nucleic acid P is ready for hybridization while being held on the beads 21, and undergoes hybridization with molecules X of a complementary nucleic acid (which will hereinafter be simply called “the target nucleic acid X”) in a sample solution supplied into the hybrid-forming section 111. The manner of hybridization is illustrated in
In
Referring next to
The beads 22 are provided with molecules L of a linker (which will hereinafter be simply called “the linker L”), which is a nucleic acid bound on the surfaces of the beads 22, for example, via avidin-biotin bonds or through a coupling reaction (for example, a diazo coupling reaction). No particular limitation is imposed on the linker L insofar as it contains a base sequence capable of undergoing complementary bonding with molecules X of a target nucleic acid (which will hereinafter be simply called “the target nucleic acid X”). A preferred example of the linker L is one containing poly(T) of the same base sequence.
With reference to
In the hybrid-forming section 111, the target nucleic acid X is ready for hybridization while being held on the beads 22 via the linker L, and undergoes hybridization with molecules P of a complementary probe nucleic acid (which will hereinafter be simply called “the probe nucleic acid P”) in the sample solution supplied into the hybrid-forming section 111. The manner of hybridization is illustrated in
In
As depicted in
By holding, for example, an mRNA extracted from cells of a subject or the like on beads 22 via the linker L, such a hybridization assay can be used in a test or the like intended to determine whether or not the probe nucleic acid P having a base sequence associated with a known gene responsible for the development of a disease undergoes hybridization with the mRNA.
The above-described assay can be performed, for example, based on a process shown in the flow sheet of
In step 2 (
Toward the hybrid-forming section 111 with the beads 22 packed therein, a first sample solution S1 which contains the target nucleic acid P is then supplied in step 3 (
A second sample solution S2 with the probe nucleic acid P contained therein is next supplied toward the hybrid-forming section 111 in step 5 (
In Example 1, an investigation was made on the effects of the length of a bead bed packed in a hybrid-forming section, which formed a column layer in a microflow path system according to an embodiment of the present invention.
As a capillary for the construction of the microflow path system, a fused silica capillary tube of 0.53 mm in inner diameter, 0.68 mm in outer diameter and 6 cm in length (product of GL Sciences, Inc.) was provided firstly.
An outlet portion fitted with a filter of 1-μm pore size and an inlet portion not fitted with any filter are attached to a downstream-side end portion and upstream-side end portion of the fused silica capillary tube by using tubing sleeves, ferrules and nuts, respectively. To the inlet portion on the upstream side, a fill port corresponding to a rheodyne syringe was fitted, and to the outlet portion on the downstream side, a luer lock needle was fitted.
As perfusion chromatography particles, “POROS 20 R1” (commercial name, product of Applied Biosystems) was used. It was dispersed in a 10% ethanol solution to prepare a particle dispersion (hereinafter referred to as “the POROS dispersion”).
A polynucleotide (21 mer) of deoxythymidine (dT) modified with biotin at the 5′ end thereof was added to an aqueous dispersion of “STREPTAVIDIN COATED MICROSPHERE PLAIN” (trade name, diameter: 6 μm, product of Polysciences, Inc.) to prepare beads with poly(dT) immobilized thereon via avidin-biotin bonds (hereinafter called “the poly(dT) beads”).
A syringe is attached to the luer lock needle on the outlet portion of the microcolumn. The rheodyne syringe with the “POROS dispersion” drawn therein was attached to the fill port at the inlet portion of the microcolumn. The plunger of the syringe fitted to the outlet portion was then pulled to inject the “POROS particles” as perfusion chromatography particles into the tube.
The injection amount of POROS was adjusted around 1.25 mg to fabricate a microcolumn having a bed of POROS alone. Ultrapure water was used as a fluid to be supplied, and was supplied up to a flow rate of 100 μL/min by successively raising the flow rate from 5 μL/min. On the downstream side, a drain flowed out corresponding to the respective flow rates. Leakage or the like was not observed.
Subsequently, the poly(dT) beads (1.3 mg) prepared by the above-described procedure were injected in a similar manner into the upstream side of the fluid-supply promoting section. The resulting bead bed length was 4 mm. By decreasing the injection amount of the poly(dT) beads, a microcolumn of 2-mm bead bed length was also formed.
Ultrapure water was supplied into the hybrid-forming section in a similar manner as in the case of the fluid-supply promoting section. Up to 100 μL/min, a drain flowed out corresponding to the respective flow rates. Leakage or the like was not observed.
From the microcolumn fabricated as described above, the luer lock needle and fill port were detached. The microcolumn was then fixed on a heated plate as a stage in a fluorescence microscope. Provided are a capillary tube with a ferrule and nut attached on only one end thereof, another capillary tube with a ferrule and nut attached on each end thereof, a syringe pump, and a drain bottle.
Firstly, the capillary tube having the ferrule and nut on each end thereof was attached to the upstream-side inlet portion of the microcolumn. The ferrule and nut on the opposite side of the capillary tube were attached to the luer lock needle via an internal union, and were connected to the syringe to be set on the syringe pump. The capillary tube with the ferrule and nut attached on only one end thereof was next attached to the downstream side of the microcolumn, and the opposite side of the capillary tube was introduced into the drain bottle.
The microcolumn set as described above was checked for leakage or the like during a fluid supply. Ultrapure water was supplied into the microcolumn, and the microcolumn was checked to determine any leakage. As a result, no leakage was determined.
Using a “RNEASY PROTECT KIT” (trade name, product of Qiagen NV), a standard total RNA kit, total RNA was extracted with ultrapure water from HeLa cells cultured in a 10-cm dish to obtain an aqueous solution of total RNA.
An aqueous solution of sodium chloride was added to the above-obtained aqueous solution of total RNA such that the concentration of sodium chloride reached a final concentration of 0.5 M.
Provided were centrifugal filter columns the filter pore sizes of which were 0.1 μm, 0.22 μm, 0.45 μm, 0.65 μm and 5 μm, respectively (“AMICON ULTRA MC” (trade name), products of Nihon Millipore K.K.). Centrifugal filtration was performed by using the filters in the decreasing order of filter pore size, and the final filtrate was provided as a sample solution.
A 0.5 M aqueous solution of sodium chloride was passed through the microcolumn to replace the internal liquid phase of the microcolumn with the 0.5 M aqueous solution of sodium chloride. The above-prepared sample solution was collected in the syringe, and the syringe was connected to the luer lock needle on the capillary tube on the upstream side of the column. By the syringe pump, the sample solution was supplied as much as 300 μL to form a hybrid between poly(dT) and the poly(A) site in the filtered sample of total RNA. Subsequent to the supply of the sample solution, a 0.1% solution of sodium dodecylsulfate (SDS) in a 0.5 M aqueous solution of sodium chloride (700 μL) was supplied to wash the inside of the microcolumn.
Employed as a probe for β-actin was a polydeoxynucleotide, which had a sequence complementary to the sequence of the 21 bases close to the poly(A) tail extension of an mRNA and was labeled on the 5′ side thereof with Cy3. For the determination of the sequence, DNA Data Bank of Japan (DDBJ) was used. The polydeoxynucleotide was dissolved in a 0.5 M aqueous solution of sodium chloride to prepare a 5 μM probe solution. The probe solution was collected in the syringe, and was supplied as much as 300 μL into the microcolumn in a similar manner as described above. Subsequent to the supply of the probe solution, a 0.1% solution of sodium dodecylsulfate (SDS) in a 0.5 M aqueous solution of sodium chloride (700 μL) was supplied to wash the inside of the microcolumn.
Fluorescence from Cy3 was measured by using a microspectroscopy system (manufactured by Otsuka Electronics Co., Ltd.) connected to a fluorescence microscope (manufactured by Nikon Instech Co., Ltd.) equipped with a Cy3 fluorescence filter, and was recorded as an amount of the complex formed. To eliminate effects of 0.1% sodium dodecylsulfate (SDS), a measurement was conducted by replacing the liquid phase with a 0.5 M aqueous solution of sodium chloride. The results of the measurement when the bead bed length of the poly(dT) beads in the hybrid-forming section was 4 mm are shown in
It has been ascertained from Example 1 that the length of a bead bed packed in a hybrid-forming section formed in a column layer of a microflow path system according to an embodiment of the present invention may desirably be set at 4 mm or shorter, with 2 mm or shorter being more preferred. The internal pressure of a microflow path drops as the bead bed length becomes shorter. Further, the bead bed length is considered to give no problem or inconvenience to fluorometric measurements insofar as it is at least as long as the inner diameter of the microcolumn.
In Example 2, an investigation was made for a suitable filter pore size upon filtering a total RNA sample solution before it is passed through a microflow path system according to an embodiment of the present invention.
Similarly to Example 1, centrifugal filter columns the filter pore sizes of which were 0.1 μm, 0.22 μm, 0.45 μm, 0.65 μm and 5 μm, respectively (“AMICON ULTRA MC” (trade name), products of Nihon Millipore K.K.) were provided. When a total RNA sample solution prepared by a similar procedure as in Example 1 was subjected to centrifugal filtration by using the filters in the decreasing order of pore size, a residual solution was observed on each of the filters. In particular, the residual solution on the 0.65 μm filter was substantial. The final filtrate was provided as a filtered sample solution. As a control for comparison, the sample solution was used without filtration.
A leakage status was: observed when the filtered sample solution was supplied into the microcolumn set by a similar procedure as in Example 1 and also when the sample solution was supplied without filtration. The bead bed length of poly(dT) beads was set at 4 mm. The results of a leakage status against flow rate are shown in Table 1, in which “A”, “B” and “C” indicate as follows. A: No leakage took place, B: Slight leakage took place although a fluid supply was feasible, and C: Leakage took place.
As shown in Table 1, it is appreciated that the filtered sample solution permitted its supply at a flow rate of 10 μL/min or higher although it occasionally developed slight leakage at the high flow rate of 50 μL/min. The sample solution without filtration, on the other hand, frequently developed leakage at connected parts or the like of the tubing when supplied at the high flow rate of 50 μL/min. A reduction in flow rate made it possible to perform its supply. To perform its supply without leakage, however, it was demanded to lower the flow rate beyond 5 μL/min to 2 μL/min in some instances.
It has been found from Example 2 that the filtration of a sample solution before its supply makes it possible to remove suspended substances from the sample solution and to prevent the clogging of a flow path. In the course of the centrifugal filtration of the sample solution, there was a substantial residual solution on the filter of 0.65 μm in pore size. It has, therefore, been found that the suitable pore size of the filter is 0.65 μm or smaller, with 0.1 μm or smaller being more preferred. The development of the slight leakage when the filtered sample solution was supplied at 50 μL/min may be considered to be attributable to a rise in internal pressure as a result of increased resistance in flow paths between beads by the mRNA subjected to poly(A) selection on the poly(dT) beads.
In Example 3, an investigation was made for possible leakage when the sample solution was supplied while performing depressurization on a downstream of a microflow path system according to an embodiment of the present invention.
By a similar procedure as in Example 1, a microcolumn was set, and a capillary tube with a ferrule and nut carried on the side of each end thereof was attached to the downstream side of the microcolumn. The downstream side of the capillary tube was connected to a syringe via an internal union and a luer lock needle.
The plunger of the syringe attached as described above was pulled. While maintaining the outlet side under a negative pressure, the sample solution was supplied. The bead bed length of poly(dT) beads was set at 4 mm. The results are shown in Table 2 in which similarly to Example 2, “A”, “B” and “C” indicate as follows. A: No leakage took place, B: Slight leakage took place although a fluid supply was feasible, and C: Leakage took place. It has been found that as shown in Table 2, no leakage took place at the flow rates of 2, 5, 10 and 20 μL/min when the filtered sample solution was supplied, and a fluid supply at 20 μL/min or higher is feasible. When the sample solution was supplied without filtration, an improvement was also observed by pulling the plunger although the improvement was slight.
It has been found from Example 3 that a fluid supply at a still higher flow rate is feasible by depressurizing the downstream side of the microflow path system according to an embodiment of the present invention. This may be attributed presumably to an improvement in a flow of liquid phase as a result of the maintenance of a negative pressure on the downstream side although a pressure is built up on the upstream side by a fluid supply from an upstream.
It is to be noted that, when the suited length (2 mm) of the bead bed length as investigated in Example 1 and the depressurization in this example were used in combination, a tendency toward a further improved fluid supply was observed.
With the microflow path system according to an embodiment of the present invention, a high flow-rate fluid supply is feasible because it can prevent clogging in a microflow path and a rise in the internal pressure of the microflow path. The microflow path system according to an embodiment of the present invention can also be used effectively for supplying a total RNA sample which contains various suspended substances, long-chain nucleic acids and the like. As the formation of a hybrid is effected on the surfaces of beads packed within the microflow path, the efficiency of the reaction is high. By washing or otherwise cleaning the beads, the microflow path system according to an embodiment of the present invention can be used as a repeatedly-usable hybridization detector.
It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and alterations may occur depending on design requirements and other factors insofar as they are within the scope of the appended claims or the equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| P2006-141896 | May 2006 | JP | national |
