Patent Application
20080014581
These and other features, objects and advantages of the present invention will become more apparent from the following description when taken in conjunction with the accompanying drawings wherein:
First, a method of manufacturing the biosensor element will be described.
The method of manufacturing the biosensor element of the present embodiment includes the following steps 1 to 7:
1. Step for cleaning carrier;
2. Step for introducing active group onto carrier surface;
3. Step for immobilizing metal particles;
4. Step for blocking carrier surface;
5. Step for immobilizing probe molecules on metal particles;
6. Step for blocking on metal particles; and
7. Step for controlling structures of probe molecules.
Now, the respective steps will be described below.
A carrier corresponding to a purpose is prepared, and subjected to cleaning. To be more precise, the carrier is cleaned with an alkaline aqueous solution such as a NaOH aqueous solution, and then cleaned with an acidic aqueous solution such as an HCl aqueous solution. The carrier is rinsed with purified water, and thereafter is subjected to drying under reduced pressure. Alternatively, organic contamination is rinsed off with a solution formed by blending sulfuric acid and hydrogen peroxide at an approximate proportion of 4 to 1.
As for the carrier, it is possible to use a glass substrate (a glass slide), a quartz substrate, a plastic substrate or the like. It is also possible to apply a metal-coated substrate, for example. It is preferable that the material of the carrier have a silanol group on a surface thereof. The carrier does not have to be of a flat form. For example, it is also possible to use the carrier of a bead form, a fiber form, a powder form or the like. In a case of the bead form, it is possible to use a carrier of a plastic bead such as polystyrene, a metal-coated bead, a magnetic bead or the like.
A silane coupling agent containing a reaction-active group is brought into a reaction with the surface of the cleaned carrier, thereby immobilizing the active group on the surface of the carrier.
As for the silane coupling agent, it is possible to use 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltrimethoxysilane or (aminoethyl-aminomethyl) phenethyltrimethoxysilane, for example, in a case of immobilizing an amino group on the surface of the substrate. Meanwhile, it is possible to use 3-mercaptopropyltrimethoxysilane in a case of immobilizing a thiol group on the surface of the substrate.
As for a solvent, it is possible to use ethanol, methanol, toluene, benzene or water, for example. A reaction temperature is usually set in a range from 20° C. to 85° C.
The metal particles are immobilized on the surface of the carrier by means of an interaction between the active groups and the metal particles on the surface of the carrier.
As for the material of the metal particles, it is possible to use any of noble metal having fluorescence-quenching and fluorescence-enhancement effects including any of gold, silver, platinum, palladium, rhodium, iridium, ruthenium, and osmium, or alloy thereof. Alternatively, it is possible to use a metal particle which is made by these noble metals and which is coated with a noble metal with another kind such as a gold particle coated with silver. The diameter of the metal particles is set equal to or above 0.6 nm so as to permit continued existence of the metal particles and equal to or below 1 μm so as to achieve a fluorescence-enhancement effect attributable to localized plasmon resonance.
Water, ethanol or toluene can be used as a reaction solvent for immobilizing the metal particles. A protective agent is used to avoid aggregation of the metal particles in the solution. As for the protective agent, it is possible to use citric acid, mercaptosuccinic acid, polyvinylpyrrolidone, polyacrylic acid, tetramethylammonium, polyethyleneimine, 1-decanethiol, 1-octanethiol or decylamine, for example.
The concentration of the metal particles is usually set equal to or below 30 wt %. The reaction temperature is usually set in the range from 20° C. to 85° C. Meanwhile, reaction time is set in a range from 0.5 hours to 50 hours. It is possible to control immobilization density of the metal particles by changing these conditions of reaction. For example, the immobilization reaction of the metal particles is a primary reaction for the concentration of the metal particles in an immobilizing solution, which is equivalent to a Langmuir-type reaction. Accordingly, it is possible to achieve desirable immobilization density by changing the concentration of the metal particles in the immobilizing solution or changing the reaction time. The immobilization density has to be equal to or above 1 particle/μm2 equivalent to the metal particle density that allows detection of the fluorescent intensity. In the meantime, the immobilization density is set preferably equal to or below 106 particles/μm2 equivalent to the metal particle density for achieving single-layer saturated immobilization.
It is also possible to form and immobilize the metal particles on the surface of the carrier by use of a combination of techniques of vapor deposition, sputtering, chemical vapor deposition (CVD), annealing, and the like. In this case, the size and density of the metal particles to be immobilized on the carrier are determined by the conditions of vapor deposition, sputtering, CVD, and annealing. For example, it is possible to control the size and the density by changing a vapor deposition temperature, a duration of vapor deposition, a pressure during vapor deposition, an amount of vapor deposition, a duration of sputtering, a gas source used for CVD, a pressure and a temperature when performing CVD, a duration of CVD, an annealing temperature, a duration of annealing, and so forth. In order to improve adhesion between the carrier and the metal particles, it is also possible to interpose a spacer such as a chromium thin film between the carrier and the metal particles.
The diameter of the metal particle stated herein means not only the diameter of a spherical particle but also the diameter in a minor axis direction of a spheroidal or columnar particle.
Among the active groups formed on the surface of the carrier, an active group that remains on the surface without immobilizing the metal particle thereon is apt to adsorb a biomolecule in a later process. For this reason, such a remaining active group needs to be blocked.
For example, a molecule containing a polyethylene glycol chain is used as the blocking molecule. As for the molecule containing the polyethylene glycol, it is possible to use carboxyl polyethylene glycol having an N-hydroxyl succinimide (NHS) activated ester at a terminal. As the binding form of the carboxyl group, it is possible to apply any of succinate, glutalate, carboxylmethyl, and carboxylpentyl. Alternatively, it is also possible to use aldehyde polyethylene glycol having an aldehyde group at a terminal. The polyethylene glycol molecules used herein typically have molecular weights equal to or below 10000.
It is also possible to suppress adsorption of the biomolecules by using bovine serum albumin (BSA) or phospholipid polymer as the other blocking molecule. However, in a case of using the aforementioned polyethylene glycol, it is possible to immobilize the blocking molecule on the surface of the substrate by covalent binding, and thereby to form a stable blocking layer. Moreover, the polyethylene glycol has a significant effect of suppressing adsorption of the biomolecules such as nucleic acids or proteins.
Now, an example will be described below in a case where the active group on the surface of the substrate is an amino group and where carboxylmethyl polyethylene glycol (molecular weight equal to 2000) containing the NHS activated ester is used as the blocking molecule.
In this case, the amino group and the NHS activated ester group are brought into a reaction. As disclosed in Bioconjugate Techniques, Elsevier Science, p. 140 (1996), the NHS activated ester group is easily hydrolyzed with an alkaline solution. On the other hand, in order to cause the reaction between the amino group and the NHS activated ester, it is essential to cause the reaction in the pH range where the amino group is not protonated, i.e., in the alkaline solution. For this reason, it has been important to find out an appropriate pH range suitable for a polyethylene glycol reaction solution.
Here, a triethanolamine solution, hydrochloric acid, a sodium carbonate solution or a sodium bicarbonate solution can be used as a pH adjuster for the polyethylene glycol solution. The reaction temperature is usually set in a range from 4° C. to 35° C. The concentration of the polyethylene glycol reaction solution is usually set in a range from 1 mM to 100 mM, and the reaction time is set in range from 10 minutes to 120 minutes.
The probe molecule having a group that can bind to the metal particles are brought onto the metal particles immobilized on the surface of the carrier to induce a reaction, thereby immobilizing the probe molecules on the metal particles.
Now, described will be a case of using a gold nanoparticle as the metal particle while using probe DNA having a thiol group at the 5′ terminal thereof as a probe molecule. The 3′ terminal of this probe DNA is modified with a fluorescent molecule. In general, when a linear distance between the metal particle and the fluorescent molecule is set equal to or below 5 nm, a speed of transfer of excitation energy from the fluorescent molecule to the metal particle is considerably increased. Thus, it is possible to quench the fluorescence. The transfer speed of the excitation energy from the fluorescent molecule to the metal particle is in inverse proportion to the sixth power of the distance between the metal particle and the fluorescent molecule. For this reason, a quenching effect becomes greater as the distance between these substances gets closer.
In a case of using the quenching probe DNA, it is possible to quench the fluorescence efficiently if an immobilized probe DNA 502 has a hairpin structure as illustrated in
Here, a “fluorescent material” means a substance that emits fluorescence when energy is applied thereto. The fluorescent material may be a cyanine dye such as Cy3 or Cy5, a rhodamine dye, a fluorescein dye or a material doped with erbium ions, for example. However, the fluorescent material will not be limited only to the foregoing substances. A single piece of the fluorescent material may be bonded to a single probe molecule. Otherwise, multiple pieces of the fluorescent material may be bonded to a single probe molecule. The fluorescent material may be bonded covalently to the probe molecule or by way of hydrogen bonding, coordinate bonding or ion bonding. Alternatively, the fluorescent material may be adsorbed to the probe molecule by way of physical adsorption.
Now, the immobilized probe DNA has a sequence in which 5 bases at both terminals are mutually complementary, such as:
In this respect, the “complementary sequence” means a sequence which can form a stable pair by way of hydrogen bonding. Specifically, the complementary sequence is based on the complementary base pairs of A to T or T to A, and C to G or G to C.
In a case where both of the terminals include the mutually complementary sequences, these two terminals collectively form hydrogen bondings 504. Accordingly, it is easy to form the hairpin structure. The length of the complementary sequences from both of the terminals is set preferably equal to or below 8 bases. In a case where the length of the complementary sequences exceeds 8 bases, the hydrogen bondings at the terminals may be strong enough to form a stable structure. As a result, it may be difficult to cause this probe DNA to react with the biomolecule in the specimen.
Meanwhile, the quenching effect can be obtained even if the number of the complementary sequences at both of the terminals is equal to 0, because the fluorescence is quenched as the fluorescent molecule is adsorbed by the metal particle even in a case where the hairpin structure is not formed by the complementary bonding of the bases in the probe DNA. Of the above-mentioned sequence of the probe DNA, a sequence of a poly-A portion which is a consecutive A-base sequence can be changed depending on a sequence of detection target DNA.
For example, in a case where double-stranded DNA is formed as a result of the reaction between this probe DNA and the biomolecule for detection, the distance between the gold nanoparticle and the fluorescent molecule becomes equal to or longer than 5 nm. As described above, since the fluorescence-quenching effect by the metal particle is in inverse proportion to the sixth power of the distance between the metal particle and the fluorescent molecule, the light emission from the fluorescent molecule can be observed in a case where the distance is increased. A fluorescence-enhancement effect attributable to the metal particle is obtained in the range of the distance from about 5 nm to about 100 nm. In this respect, a relationship between the distance in the range from about 5 nm to about 100 nm and a proportion of the fluorescence-enhancement is determined by the type of the fluorescent molecule used therein as well as by the type and size of the metal particle. In every system, the fluorescence-enhancement is observed in the range from about 5 nm to about 100 nm. However, there is a certain distance in the range from about 5 nm and about 100 nm where the highest enhancement effect is obtained.
The fluorescence is enhanced in a case where the distance of between the metal particle and the fluorescent molecule modified on the probe molecule is set in the range from about 5 nm to about 100 nm when the biomolecule reacts with the probe molecule. When one of the terminal of the probe DNA is immobilized on the gold nanoparticle while the other terminal thereof is modified with the fluorescent molecule, it is preferable to set the length of the probe DNA in the range from about 5 nm to about 100 nm.
An almost neutral aqueous solution such as a phosphate buffer is applicable as a solution for dissolving the probe DNA. The probe DNA is dissolved in this solution. The concentration of the probe DNA at this time is usually set in a range from 0.5 μM to 100 μm.
In a case where the carrier is made of a flat glass substrate and the gold nanoparticle is immobilized thereon, a reaction solution dissolving the probe DNA may be spotted in desired positions on the substrate. At this time, it is possible to spot multiple types of the probe DNA onto the substrate. In a case where the carrier is made of a bead, the bead may be immersed in the reaction solution dissolving the probe DNA.
The reaction temperature is usually set in a range from 25° C. to 40° C. Meanwhile, the reaction time is usually set in a range from 2 hours to 24 hours. In order to prevent the solution from drying during the reaction, the reaction should be taken place in an environment where a humidity is properly maintained.
Since the gold easily binds to the thiol group, the probe DNA having the thiol group at the terminal is immobilized solely on the metal particle. A portion where no gold nanoparticle is immobilized is covered with polyethylene glycol. Accordingly, the probe DNA is hardly adsorbed on that portion.
In this example, the metal particles are firstly immobilized on the substrate. Then the surface of the substrate where the metal particles are not immobilized is subject to blocking. Thereafter, the probe molecules are immobilized thereon. Instead, it is also possible to firstly immobilize the probe molecules on the metal particles, then to immobilize the metal particles on the surface, and then to block the surface of the substrate where the metal particles are not immobilized.
A portion on the surface of the metal particle where the probe DNA is not immobilized may adsorb the biomolecule for detection. For this reason, the remaining surface of the metal particle is subjected to blocking.
Described will be a case of using the gold nanoparticles as the metal particles. It is possible to use 1-mercaptohexanol, 2-mercaptoethanol, or the like as a blocking agent that reacts easily with gold and hardly adsorbs the biomolecule. An aqueous solution of 1-mercaptohexanol or 2-mercaptethanol is reacted with the surface of the carrier so as to immobilize the blocking material.
The reaction temperature is usually set in a range from 4° C. to 35° C. Meanwhile, the reaction time is usually set in a range from 0.5 hours to 10 hours. In this reaction, in a case where the concentration of the blocking agent in the aqueous solution is high, the blocking agent reacts with the gold nanoparticles, and thereby covers the gold nanoparticles. In this way, the blocking agent weakens the binding force between the carrier and the nanoparticle. As a result, the metal particles are dispersed on the surface of the carrier, and precipitate on the surface. A relationship between the concentration of the blocking agent and the presence of the surface dispersion is shown in Table 1. Based on Table 1, the concentration of the blocking agent reaction solution is set equal to or below 100 μM.
The step 5 and the step 6 are conducted separately. However, it is also possible to conduct the steps 5 and 6 at the same time. Specifically, when immobilizing the probe molecules on the metal particles, it is possible to carry out immobilization at the same time by use of a solution dissolving the probe molecules as well as the blocking agent for the metal particles.
The structures of the probe molecules immobilized on the metal particles are controlled such that the fluorescence is efficiently quenched by the metal particles. As previously described in the step 5, the fluorescent molecule placed in the vicinity of the metal causes excitation energy of the fluorescent molecule to transfer to free electrons in the metal, and thereby quenches the fluorescence. The quenching effect is high in a case where the distance between the metal particle and the fluorescent molecule is extremely small. In contrast, the quenching effect is substantially reduced in a case where the distance is increased. In order to render the distance between the fluorescent molecule and the metal particle as small as possible and thereby to quench the fluorescence efficiently, the hairpin structure as shown in
Described will be a case of using the gold nanoparticles as the metal particles 501 while using the probe DNA as the probe molecules 502. When the surface of the substrate, on which a probe DNA is placed, is exposed to a solution having low basic strength, the negatively charged bases by way of phosphate bases in the DNA repel each other. Accordingly, the probe DNA is stretched, and is hardly formed into the hairpin structure. It is necessary to form the hairpin structure, therefore, in a solution having appropriate ionic strength.
As this solution, it is possible to use sodium carbonate, potassium carbonate, sodium phosphate, potassium phosphate, magnesium chloride, and the like. The ionic strength of the solution is usually set in a range from 50 mM to 2 M. The temperature is usually set in a range from 25° C. to 45° C., and the reaction time is usually set in a range from 0.5 hours to 5 hours. Thereafter, the substrate is taken out of the solution and dried. Alternatively, it is possible to preserve the substrate while keeping contact with the above-described ionic solution.
Next, a detection method using the biosensor element manufactured in accordance with the above-described steps 1 to 7 will be described below.
Excitation light is irradiated on a surface of the biosensor element manufactured in accordance with the above-described steps 1 to 7 by use of a fluorescent scanner so as to detect light emission from the surface. If the distance between the fluorescent molecule modified at the terminal of the probe molecule and the metal particle is small enough, the fluorescence is quenched, and detected fluorescent intensity is extremely small.
Next, a biomolecule for detection and the above-described biosensor element are brought into a reaction. To be more precise, the surface of the biosensor element is allowed to contact the solution dissolving the biomolecule for detection and to continue the reaction until reaching the equilibrium. Described will be a case of using the probe DNA as the probe molecule while using the nucleic acid as the biomolecule for detection.
The nucleic acid for detection is dissolved in a surfactant-added standard saline citrate (SSC) solution, and this solution is allowed to contact the surface of the biosensor element. An amount of the nucleic acid in the solution is set in a range from 0.1 amol to 1 nmol. The reaction temperature is usually set in a range from 25° C. to 60° C., and the reaction time is usually set in a range from 1 hour to 24 hours. In a case where the nucleic acid for detection is completely complementary to the sequence of the probe DNA, the nucleic acid reacts quickly to form double-stranded DNA that is linked by way of hydrogen bonding. The double-stranded DNA considerably loses flexibility as a polymer, and becomes like a rigid spring. After the reaction, a structural change occurs from a state illustrated in
When the light is irradiated on the metal particle, the free electrons in the metal particle are polarized and oscillated. Resonance between the oscillation of the free electrons in the metal particle and an oscillating magnetic field attributable to the incident light is called as the “localized plasmon resonance.” When the localized plasmon resonance is generated, electric field intensity on the surface of the metal particle is increased by several digits as compared to the electric field intensity attributable to the incident light.
Next, two factors for the above-described fluorescence-enhancement will be described. A first factor for the fluorescence-enhancement is attributed to improvement in quantum efficiency of the fluorescent molecule. In a case where the metal particle is present in the vicinity of the fluorescent molecule, absorption transition occurs in the vicinity of the metal particle during the process of absorbing energy of the fluorescent molecule. The transition is attributable to an electric field enhancing effect owing to the localized plasmon resonance. In addition, the presence of the metal particle causes new light emission in a light emitting process.
Therefore, the quantum efficiency of the fluorescent molecule is enhanced by the absorption transition and the increase in the light emission. However, since the quantum efficiency never exceeds 1, the increase in the quantum efficiency attributable to the metal particle cannot be expected in a case of the fluorescent molecule that has the quantum efficiency equal to 1. Nevertheless, the fluorescent molecules used in the biosensor typically have the quantum efficiency in a range from about 0.04 to 0.3. Therefore, it is possible to expect the improvement in the quantum efficiency attributable to the metal particles when using these fluorescent molecules.
A second factor is an increase in light scattering intensity attributed to the metal particle. When polarizability of the metal particle is increased by the localized plasmon resonance and the electric field is enhanced in its vicinity, the light scattering intensity from the metal particle is also enhanced. This is attributed to the fact that the light scattering intensity is in proportion to the square of the polarizability of the metal particle. Along the increase in the light scattering intensity, incident energy for exciting the fluorescent molecule is also increased. Accordingly, fluorescence emission intensity is enhanced as well.
These fluorescence-enhancement effects are observed when the distance between the metal particle and the fluorescent molecule is in the range from 5 nm to 100 nm. In a case where the distance (d2) along the probe molecule between the end of the probe molecule immobilized on the metal particle and the fluorescent molecule modified on the probe molecule is in the range from 5 nm to 100 nm equivalent to the length where the fluorescence-enhancement effect is available, it is possible to utilize the fluorescence-enhancement effect after the probe molecule reacts with a specimen molecule. Accordingly, it is possible to detect the biomolecule in the specimen at ultrahigh sensitivity without labeling by highly efficient fluorescence-quenching and fluorescence-enhancement effects using the metal particles.
In accordance with this principle, in a case where the probe molecule 602 shown in
In a case where the biosensor element formed by spot-immobilizing the multiple types of the probe DNA is used for measuring the intensity of fluorescent from the surface of the substrate after causing the biomolecules in the specimen to react with the probe DNA on the spots, fluorescence may be quenched on a certain spot whereas strong fluorescent intensity may be detected on another spot. From this result, it is apparent that the specimen does not contain the biomolecule related to the probe sequence on the spot where the fluorescence is quenched, and that the specimen contains the biomolecules related to the probe sequence on the spot where the strong fluorescent intensity is detected. Moreover, it is possible to quantitatively calculate the amount of the existing biomolecules by use of the measured fluorescent intensity.
Examples of the method of manufacturing the biosensor element of the present invention and the method of detecting a biomolecule using the element have been described. Although the embodiment has described the case of applying DNA as the biomolecule, it is also possible to apply other biomolecules such as RNA, proteins, PNA, sugar chains or composites thereof.
By using the biosensor element of the present invention, it is possible to detect the specimen molecules with excellent repeatability without amplifying and labeling them. Moreover, it is also possible to perform a quantitative analysis of an amount of gene expression, a highly selective analysis of SNPs, a highly selective analysis of proteins, or the like by use of the biosensor element of the present invention.
Next, the present invention will be described more in details with reference to examples. It is to be noted, however, that the following examples will not limit the scope of the present invention. The examples to be described below are based on the cases of applying the present invention to a flat-plate DNA microarray and to a bead array.
A glass slide made of borosilicate glass is prepared as a carrier. The substrate is cleaned with an NaOH aqueous solution, then cleaned with an HCl aqueous solution, then rinsed with purified water. Thereafter, it is subjected to drying under reduced pressure. As shown in
Next, a citric acid solution containing gold nanoparticles in a diameter of 15 nm is brought onto the aminated substrate to effect a reaction. Note that, the concentration of the gold nanoparticles is set equal to 0.007% (weight/volume). Meanwhile, the reaction temperature is set to room temperature, and the reaction time is set equal to 20 hours. In this way, the substrate on which gold nanoparticles 301 are dispersed and immobilized was obtained as shown in
(Step 2) Immobilization of Blocking Agent
Triethyl alcohol (TEA) in a concentration of 100 mM is adjusted to pH 8.0 by use of an HCl solution, and polyethylene glycol chains at a molecular weight of 2000 having succineimide-activated ester at terminals are dissolved in the solution. The multiple substrates on which the gold nanoparticles are immobilized as described above are immersed in the solution immediately after dissolving the polyethylene glycol chains. The reaction temperature is set equal to 25° C., and the reaction time is set equal to 1 hour. After the reaction, the substrates are cleaned with purified water, and subjected to drying under reduced pressure.
In this way, the substrate shown in
5 μM probe DNA having a base sequence of 30 to 60 pieces long and provided with a thiol group at the 5′ terminal as well as Cy3 functioning as the fluorescent molecule at the 3′ terminal was dissolved in a weak acidic phosphate buffer adjusted to pH 6.7 by mixing 50 mM of K2HPO4 with 50 mM of KH2PO4. The probe DNA dissolving solution was spotted at every probe DNA sequence onto the substrate which was subjected to blocking as described above. In this way, the substrate immobilizing multiple types of the probe DNA 302 thereon was obtained as shown in
Note that, the reaction temperature is set equal to 25° C., and the reaction time is set equal to 4 hours. Moreover, in order to prevent the solution from drying during the reaction, the reaction was conducted in an environment where a humidity was properly maintained. After the reaction, the substrate was cleaned with purified water.
A mercaptohexanol aqueous solution in a concentration of 1 μM was prepared, and the substrate immobilizing the probe DNA was immersed in this aqueous solution. The reaction temperature is set equal to 25° C., and the reaction time is set equal to 1 hour. After the reaction, the substrate was cleaned with purified water, and then subjected to drying under reduced pressure in a desiccator. In this way, the substrate shown in
The surface of the substrate after blocking the surfaces of the gold nanoparticles as described above was immersed in a 2×SSC solution having basic strength of 0.3 M. The immersing temperature is set equal to 25° C., and the immersing time is set equal to 2 hours. Thereafter, the substrate was taken out of the solution and subjected to drying under reduced pressure. In the meantime, the 2×SSC solution was spotted on the same type of the substrate. By covering with a glass cover, the solution was allowed to contact the entire surface of the substrate. The substrate was preserved at 25° C.
The fluorescent intensity from the probe DNA immobilized on the substrate was measured with the fluorescent scanner used in the step 5. A laser beam having a wavelength of 530 nm was used for scanning the surface of the substrate to excite the fluorescent dye Cy3, and strength of obtained fluorescence was measured. The results are shown in the sections titled “before hybridization” in
The surface capable of minimizing the probe DNA including nonspecifically adsorbed fluorescent molecules was constructed in accordance with the step 2. The probe DNA structures were controlled in accordance with the step 5. In this way, it was possible to suppress the fluorescence from the probe DNA, namely, the fluorescence subjected to background noises.
The substrate immobilizing the probe DNA thereon was subjected to hybridization with single-stranded target DNA having the completely complementary sequence to the probe DNA but not having a label thereon. A mixed solution of 5×SSC (standard saline citrate) and a 0.5% SDS (sodium dodecyl sulfate) solution was used as a hybridization solution, and a total amount fmol of the target DNA was hybridized at 42° C. for 4 hours. Then, the substrate was cleaned with a 2×SSC, 0.1% SDS solution and with a 2×SSC solution and then subjected to drying under reduced pressure. The excitation light was made incident on the dried surface of the substrate with the fluorescent scanner, and the fluorescent intensity from the surface was measured.
In a case where the sequences of the target DNA and the probe DNA were completely complementary to each other, the fluorescent intensity was significantly increased after hybridization. The results are shown in the sections titled “unlabeled DNA after hybridization” in
In order to manufacture a bead array for a gene analysis, beads immobilizing the probe DNA were obtained in accordance with a method similar to the step 1 to the step 4 of the example 1 by use of beads instead of the substrate. Although the probe DNA was spotted in the step 3 of the example 1, the probe DNA was immobilized on the beads by immersing the beads in a solution dissolving the probe DNA. In this case, the probe DNA having a single type of sequence was immobilized on a single bead. Hence, multiple types of the beads were obtained by immobilizing multiple types of probe DNA on the multiple beads. The bead material used herein is made of borosilicate glass, and the diameter of each bead is approximately equal to 100 μm.
When the fluorescent intensity from surfaces of the beads before hybridization was measured in accordance with the method similar to the steps 5 and 6 of the example 1, it was confirmed that the fluorescence was quenched by the fluorescence-quenching effect attributable to the gold nanoparticles. The results are shown in
Next, a sample fluid which contained target DNA having a completely complementary sequence to one of the multiple types of the probe DNA immobilized on the surfaces of the beads was poured in the microchannel 801 for the hybridization in accordance with the method of the step 7 of the example 1. Then, the fluorescent intensity and uniformity were examined. As a result, high fluorescent intensity was observed only out of the bead immobilizing the probe DNA which had the completely complementary sequence to the sequence of the target DNA as shown in
To examine a difference in a gene detecting performance relative to the variation in the shapes of the metal particles, the gold nanoparticles and the blocking agent were immobilized on SiO2 on the surface of the substrate in accordance with a method similar to the step 1 and the step 2 of the example 1. A substrate prepared by coating a gold thin film (about 50 nm) on glass and then coating SiO2 on this gold thin film by sputtering in a thickness of 10 nm was used so that the substrate that can also measure surface plasmon resonance (SPR). While the step 1 of the example 1 applied only the gold nanoparticles having a diameter of 15 nm, the gold nanoparticles in various sizes were immobilized in this example, namely, those having diameters of 5 nm, 6 nm, 10 nm, 15 nm, 30 nm, 50 nm, and 80 nm. The concentration of the gold nanoparticle citric acid solution used therein is set to the gold content of 0.01% (weight/volume) in the case of the gold nanoparticle solution for the diameter of 5 nm, 6 nm or 10 nm, and the gold content of 0.007% (weight/volume) in the case of the gold nanoparticle solution for the diameter equal to or above 15 nm.
Providing that a distance between the centers of the mutually adjacent gold nanoparticles was denoted as “L” and that the diameter of each particle was denoted as “D,” an immobilization interval between the gold nanoparticles L/D was approximately equal to 2 or greater in this case. There is a report that an adverse effect of an interaction between the particles is caused by electromagnetic fields around the particles that interfere with each other in a case where the interval L/D is close to 2 or below (“Interparticle Coupling Effects on Plasmon Resonances of Nanogold Particles,” Nano Letters Vol. 3, No. 8, p. 1087-1090 (2003), “Optical Properties of Two Interacting Gold Nanoparticles,” Optics Communications 220, p. 137-141 (2003), “Electrodynamics of noble metal nanoparticles and nanoparticle clusters,” Journal of Cluster Science Vol. 10, No. 2 (1999)).
In this example, evaluation was conducted in a region where there was little effect of the interaction between the particles by setting the interval L/D approximately equal to 2 or greater. In other words, this example applied an immobilization surface with which it was possible to evaluate the effect of the diametric size of the gold nanoparticle on the gene detection performance directly.
Next, the probe DNA was immobilized on this substrate. To be more precise, the probe DNA was immobilized by use of the probe DNA dissolving solution described in the step 3. Although the DNA having the mutually complementary sequences at both terminals thereof was used as the probe DNA in the example 1, the probe DNA used in this example had any of the following 18-mer and 50-mer sequences not having the complementary sequences at both terminals thereof.
18-mer sequence:
50-mer sequence:
Meanwhile, although the example 1 applied only Cy3 as the fluorescent molecules, this example applied two types of DNA modified with Cy3 and Cy5 at the 3′ terminals. Each type of the probe DNA has a thiol group at the 5′ terminal.
When immobilizing the probe DNA, measurement applying the surface plasmon resonance (SPR) was conducted in order to find amounts of immobilization of the probe DNA. Now, this SPR measurement method will be described below. The probe DNA immobilization is carried out by causing the reaction between the gold nanoparticles immobilized on the surface of the above-described substrate and the thiol groups at the terminals of the probe DNA. In this respect, when the light is made incident from the back side of the substrate, i.e. from the glass side, through an optical prism embedded in a SPR device, light reflectance is extremely reduced at a certain incident angle where the surface plasma oscillation on the surface of gold is induced. This angle is shifted depending on small variation in the mass (permittivity) on a surface of a sensor. Accordingly, it is possible to detect the amount of the probe DNA which is immobilized on the surface of the substrate by measuring an amount of the shift of this incident angle.
In this example, the substrates formed by immobilizing the gold nanoparticles in the sizes of 5 nm, 6 nm, 10 nm, 15 nm, 30 nm, 50 nm, and 80 nm thereon and immobilizing the blocking agent thereon were set in the SPR device. The solution containing the 18-mer probe DNA and the solution containing the 50-mer probe DNA which were adjusted to 10 μM were infused in the SPR device. Thereafter, the amounts of immobilization per unit area were calculated. Area occupancies of the probe DNA molecules on the surfaces of the gold nanoparticles, which were calculated by use of those amounts of immobilization, ranged from 1 nm2 to 4 nm2 in the case of the 18-mer probe DNA, and from 9 nm2 to 14 nm2 in the case of the 50-mer probe DNA.
Subsequently, the substrates were taken out of the SPR device, and the structures of the probe DNA immobilized on the substrates were controlled in accordance with a method similar to the step 5 of the example 1. Although the substrate was immersed in the 2×SSC solution for 2 hours in the example 1, the substrates were immersed in the 5×SSC solution for a period from 5 minutes to 2 hours in this example.
Next, the fluorescent intensity emitted from the probe DNA immobilized on the substrates was measured by use of the fluorescent scanner in accordance with a method similar to the step 6 of the example 1. Although the surface of the substrate was scanned by irradiating the laser beam having the wavelength of 530 nm in the example 1, a laser beam having a wavelength of 635 nm was used as the excitation light when applying Cy5 to the fluorescent molecules while a laser beam having a wavelength of 532 nm was used as the excitation light when applying Cy3 to the fluorescent molecules. Thereafter, the fluorescent intensity emitted from Cy3 or Cy5 was measured. In this respect, the fluorescent intensity detected in one pixel of 10 μm×10 μm was measured. This fluorescent intensity measurement was carried out while immersing the surface of each substrate in the 5×SSC solution.
The fluorescent intensity per piece of the probe DNA, i.e. the fluorescent intensity per fluorescent molecule can be calculated by use of the number of the immobilized probe DNA molecules detected in one pixel of 10 μm×10 μm obtained from a result of the above-mentioned SPR measurement and the fluorescent intensity obtained with the fluorescent scanner. Calculated values of the fluorescent intensity per fluorescent molecule are shown in
Next, single-stranded target DNA without fluorescence labeling was hybridized with the probe DNA immobilized on the above-described substrates in accordance with a method similar to the step 7 of the example 1. The sequences of the target DNA used in this example include completely complementary sequences and random sequences which do not have distinctive complementary sequences. The complementary sequences used therein are as follows.
18-mer sequence:
50-mer sequence:
Meanwhile, the random sequences used therein are as follows.
18-mer sequence:
50-mer sequence:
In the step 7 of the example 1, the cleaning and drying processes are executed after hybridization, and then the fluorescent intensity from the substrate is measured by use of the fluorescent scanner. In contrast, in this example, the substrates were cleaned with 5×SCC solution after hybridization, and then the fluorescent intensity was measured while immersing the substrates in the 5×SCC solution. A reason for conducting this operation is as follows. The fluorescent intensity varies depending on the permittivity of the solvent that surrounds the fluorescent molecule. Accordingly, in a case of comparing the fluorescent intensity between environments before and after hybridization more accurately, it is more appropriate to perform measurement while using the same solvent in both of the environments. In other words, it is more appropriate to perform measurement while immersing the substrates in the 5×SCC solution in both of the environments. In this case, it is possible to compare the fluorescent intensity between the environments before and after hybridization without considering an influence of the solvent surrounding the fluorescent molecule. Another reason is that it is possible to prevent denaturation of the DNA structures caused by the drying process. In this example, the amount of the target DNA was changed from 1 fmol to 1 pmol.
Values of average fluorescent intensity per fluorescent molecule when causing the reaction of 1 pmol of the target DNA are shown in
A result of calculated contrast C(C=I2/I1) representing a ratio between the fluorescent intensity (I1) before hybridization and the fluorescent intensity (I2) after hybridization is shown in
A relation between the size of the gold nanoparticle and the contrast will now be explained with reference to
In this respect, a reason of the higher contrast in the aforementioned range will be described below. To obtain high contrast, it is necessary to quench the fluorescence efficiently before hybridization and to enhance the fluorescence efficiently after hybridization. The degree of fluorescence-enhancement is increased along with an increase in the size of the gold nanoparticle. Since the polarizability of the metal particle is in proportion to the third power of a particle diameter, the polarizability is reduced in a case where the particle size is small, and electromagnetic strength around the particle is also reduced in this case. In a case where the particle diameter is equal to or below 5 nm, it is hard to obtain the fluorescence-enhancement effect, and is therefore hard to obtain high fluorescent intensity after the reaction with the biomolecule. In contrast, in a case where the particle diameter is equal to or above 50 nm, a strong fluorescence-enhancing field is generated around the particle. Accordingly, it is hard to cause fluorescence quenching, and high fluorescent intensity is obtained even before the reaction with the biomolecule. Thus, the ratio of the fluorescent intensity (the contrast) before and after the reaction with the biomolecule becomes small.
From this point of view, it is appropriate to set the particle diameter of the metal particle in the range from 5 nm to 50 nm inclusive in order to perform the highly sensitive measurement of the biomolecule at high contrast. To obtain even higher contrast, it is appropriate to set the particle diameter of the metal particle in the range from 6 nm to 15 nm inclusive.
Next, a relation between the length of the probe DNA and the contrast will be explained. When comparing the contrast of the 50-mer probe DNA and the contrast of the 18-mer probe DNA, it is apparent that the contrast of the 50-mer probe DNA is the higher. A reason of this aspect will be described below. As shown in
In a case where the probe DNA is 18-mer long, a chain length in a case of generating double-stranded chain is approximately equal to 6 nm. If the length is merely as long as 6 nm, the fluorescent molecule is located within the fluorescence-quenching field in proximity to the gold nanoparticle even after hybridization. Accordingly, the fluorescent molecule is not able to fully transit to the fluorescence-enhancing field where the stronger fluorescent intensity is obtainable. In contrast, in a case of the 50-mer probe DNA, a chain length of the generated double-stranded chain is approximately equal to 17 nm. Accordingly, the fluorescent molecule can transit to the fluorescence-enhancing field by forming the rigid double-stranded chain, and the contrast is enhanced as a consequence.
In this example, the substrate prepared by coating the gold thin film (about 50 nm) on the glass and then coating SiO2 on this gold thin film by sputtering in a thickness of 10 nm was used as the substrate in order to perform the SPR measurement. However, it is also possible to obtain similar results on a substrate prepared by forming a SiO2 thin film on a silicon substrate, on fused silica or on glass.
In this example, the degree of the fluorescence enhancement, in a case of using the gold nanoparticles, was examined in order to detect genes at high sensitivity. To be more precise, a relationship between the degree of the fluorescence enhancement and the diameter of the gold nanoparticle was examined by quantitatively obtaining the degree of the fluorescence enhancement. An array used in this example was fabricated in accordance with a method similar to the method described in the example 3. Specifically, a substrate was prepared by coating SiO2 on a gold thin film by sputtering in a thickness of 10 nm and then various particle diameters of gold nanoparticles and probe DNA attaching fluorescent molecules (Cy3 or Cy5) were immobilized on the substrate to fabricate the array. This example applied the probe DNA having the following sequence:
50-mer sequence:
Meanwhile, in order to quantitatively evaluate the fluorescence-enhancement effect, another array was fabricated by immobilizing the probe DNA attaching the fluorescent molecules without using the gold nanoparticles. A functional group was coated on the surface of the substrate in accordance with a method similar to the step 1 of the example 1. Although 3-aminopropyltrimethoxysilane was used in the example 1, 3-mercaptopropyltrimethoxysilane was used in this example. Meanwhile, toluene was used as a reaction solvent. Next, the probe DNA was immobilized in accordance with a method similar to the step 3 of the example 1. The probe DNA has the same sequence as the above-mentioned sequence of the probe DNA immobilized on the gold nanoparticles. In this case, the probe DNA was bonded covalently to the substrate by forming disulfide bonds. In this way, the substrate on which the probe DNA attaching the fluorescent molecules was immobilized through the gold nanoparticles as well as the substrate on which the probe DNA attaching the fluorescent molecules was similarly immobilized without the gold nanoparticles were fabricated.
Next, immobilized amounts of the probe DNA were calculated by use of the surface plasmon resonance (SPR) in a case where the substrate was provided with the gold nanoparticles and in a case where the substrate was provided without the gold nanoparticles. As described previously, in the case where the gold nanoparticles were provided, the substrate to be used was formed as follows. The gold particles had the diameters of 5 nm, 6 nm, 10 nm, 15 nm, 30 nm, 50 nm, 80 nm, 100 nm, 200 nm, 300 nm, and 500 nm, and they were immobilized on the surfaces of the substrates. Thereafter, the blocking agent was immobilized thereon. On the other hand, in the case where the gold nanoparticles were not provided, the substrate to be used was coated with the thiol groups by way of 3-mercaptopropyltrimethoxysilane. These substrates were set on the SPR device, and then the above-described 50-mer probe DNA solution adjusted to 10 μM was infused thereon in the SPR device to measure the immobilized amounts per unit area.
Subsequently, hybridization was carried out on the above-described substrate by use of the substrates immobilizing the probe DNA thereon in accordance with a method similar to the step 7 of the example 1. DNA used as the target DNA had the completely complementary sequence to the probe DNA but no label. The target DNA used in this example had the following sequence:
50-mer sequence:
In the step 7 of the example 1, the cleaning and drying processes were executed after hybridization, and the fluorescent intensity was measured thereafter. In contrast, in this example, after the substrate was cleaned, the fluorescent intensity was measured while the substrate was immersed in the 5×SCC solution. The average fluorescent intensity per fluorescent molecule was calculated from the immobilized amounts of the probe DNA attaching the fluorescent molecules which were obtained by the above-described SPR measurement and from the measured fluorescent intensity. Note that, in this example, the amount of the target DNA used for the reaction was set equal to 1 pmol.
Fluorescent intensity (I3) per fluorescent molecule after hybridization using the substrate on which the gold nanoparticles were immobilized was compared with fluorescent intensity (I4) per fluorescent molecule after hybridization using the substrate on which the gold nanoparticles were not immobilized. Here, the substrate on which the gold nanoparticles were immobilized had the higher fluorescent intensity. The results of obtaining fluorescence-enhancement coefficients E defined as E=I3/I4 are shown in
On the other hand, in a case where the diameter of the gold nanoparticle becomes almost as large as the wavelength, the polarization hardly occurs in the gold nanoparticle and the electromagnetic field around the gold nanoparticle is also reduced along with reduction in the polarizability. Therefore, it is possible to obtain a high fluorescence-enhancement effect by using the gold nanoparticles having the particle diameters in a range from 10 nm to 500 nm inclusive.
Now, comparison between Cy3 and Cy5 will be considered. In a case where the diameter of the gold nanoparticle is smaller than 100 nm, the fluorescence-enhancement coefficient of Cy3 became greater than that of Cy5. A reason for this aspect will be described below. The gold nanoparticle exerts light absorption attributable to the localized plasmon resonance by the polarization thereof. In a case where the diameter of the gold nanoparticle is smaller than 100 nm, a wavelength for this absorption is equal to somewhere from 500 nm to 550 nm. In this wavelength band, the localized plasmon resonates with the light incident on the gold nanoparticle, thereby increasing light absorption or near-field scattering. In a case where the wavelength inducing the localized plasmon resonance is used as an excitation wavelength, the strength of the near-field light scattering is increased. Thus, it is conceivable that the fluorescent intensity is enhanced more. The excitation wavelength for Cy3 ranges from 500 nm to 550 nm which coincides with the localized plasmon resonance wavelength band. In contrast, the excitation wavelength for Cy5 ranges from 600 nm to 650 nm which deviates from the resonance wavelength band. It is, therefore, conceivable that the larger fluorescence enhancement is achieved with Cy3.
In this example, the substrate prepared by coating the gold thin film (about 50 nm) on the glass and then coating SiO2 on this gold thin film by sputtering in a thickness of 10 nm was used as the substrate in order to perform the SPR measurement. However, it is also possible to obtain similar results on a substrate prepared by forming a SiO2 thin film on a silicon substrate, on fused silica or on glass.
In this example, detection was attempted while utilizing the fluorescence enhancement by the gold nanoparticles for the purpose of highly sensitive detection of fluorescence-labeled genes.
Meanwhile, in order to quantitatively evaluate the fluorescence-enhancement effect, another substrate was manufactured by immobilizing the probe DNA without using the gold nanoparticles. As similar to the example 4, the probe DNA was immobilized after coating 3-mercaptopropyltrimethoxysilane. Although the probe DNA attaching the fluorescent molecules was immobilized in the example 4, the immobilized probe DNA did not attach the fluorescent molecules but had the same sequence as the above-described probe DNA immobilized on the gold nanoparticles in this example. In this way, the substrate on which the probe DNA was immobilized through the gold nanoparticles as well as the substrate on which the probe DNA was immobilized without the gold nanoparticles were fabricated.
Next, 1 pmol of single-stranded target DNA having a completely complementary sequence to the sequence of the probe DNA was subjected to hybridization by use of the substrates on which the above-described probe DNA was immobilized. The target DNA having no label was used in the example 4. Instead, in this example, the used target DNA was modified with the fluorescent molecules (Cy3 or Cy5) functioning as the label at the 3′ terminals thereof
In order to calculate the fluorescent intensity per reacted fluorescent molecule, a reacting amount of the hybridized target DNA was calculated at the time of the hybridization described above. Upon calculation of the reacting weight, the surface plasmon resonance (SPR) was used as similar to the method described in the example 3. The substrate fabricated for SPR measurement was set on the SPR device, then a solution containing the target DNA was infused thereon in the SPR device, and then an amount of the hybridized target DNA per unit area was calculated. The fluorescent intensity per hybridized fluorescent molecule was calculated from a measurement result of this SPR hybridized amount and the above-described measurement result of the fluorescent intensity.
The fluorescent intensity (I3) per fluorescent molecule after hybridization using the substrate on which the gold nanoparticles were immobilized was compared with the fluorescent intensity (I4) per fluorescent molecule after hybridization using the substrate on which the gold nanoparticles were not immobilized. Here, the substrate on which the gold nanoparticles were immobilized had the higher fluorescent intensity. As a result of obtaining the fluorescence-enhancing coefficients E defined as E=I3/I4, it was possible to enhance the fluorescent intensity ten times or more as similar to the example 4 in a case where the diameter of the gold nanoparticle was equal to or above 10 nm. Based on the relationship between the diameter of the metal particle and the polarizability described in the example 4, it was possible to obtain a high fluorescence-enhancement effect as similar to the example 4 by using the gold nanoparticles having the particle diameters in the range from 10 nm to 500 nm inclusive.
In this example, the substrate prepared by coating the gold thin film (about 50 nm) on the glass and then coating SiO2 on this gold thin film by sputtering in a thickness of 10 nm was used as the substrate in order to perform the SPR measurement. However, it is also possible to obtain similar results on a substrate prepared by forming a SiO2 thin film on a silicon substrate, on fused silica or on glass.
Moreover, the used target DNA had the completely complementary sequence to the sequence of the probe DNA in this example. However, it is also possible to obtain similar effects when a target molecule for detection applies a single-base sequence labeled with a fluorescent molecule such as dCTP-Cy3 or ddCTP-Cy3, a protein modified with a fluorescent molecule, a carbohydrate chain or glycoprotein modified with a fluorescent molecule, and so forth.
While we have shown and described several embodiments in accordance with out invention, it should be understood that disclosed embodiments are susceptible of changes and modifications without departing from the scope of the invention. Therefore, we do not intend to be bound by the details shown and described herein but intend to cover all such changes and modifications a fall within the ambit of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-170538 | Jun 2006 | JP | national |
