WO2008030071A1

WO2008030071A1 - Detecting method of dna hybridization using scattering

Info

Publication number: WO2008030071A1
Application number: PCT/KR2007/004356
Authority: WO
Inventors: Du Soo Chung; Dai Sik Kim; Jong Won Kim; Jing Yu Piao
Original assignee: Seoul National University Industry Foundation
Priority date: 2006-09-09
Filing date: 2007-09-08
Publication date: 2008-03-13
Also published as: KR100904825B1; KR20080023278A

Abstract

The invention is about detection method of DNA hybridization on DNA chip, that is detection of DNA hybridization on DNA chip based on light scattering of probe nanoparticle DNA used in the invention. As the invention is to present new detection method of DNA hybridization on DNA chip, it can easily detect DNA hybridization on DNA chip using low cost lamp scanner without modification of biomolecules with fluorescent dye molecules or expensive optical instrument, and contribute to widely application of DNA chip.

Description

[DESCRIPTION] [Invention Title]

DETECTING METHOD OF DNA HYBRIDIZATION USING SCAHERING [Technical Field]

The invention is about detection method of DNA hybridization and synthesis method of probe nanoparticle DNA used in above detection method, in detail is that detection method of DNA hybridization on DNA chip based on detection of light scattering of probe nanoparticle DNA.

[Background Art]

There are .three kinds of biochip according to different target biomolecules, huge DNA sequence information from human genome project make it possible to find and cure a genetic disease in an early stage and give much more expectation, among them DNA chip has been most widely developed and some used commercially. For gene detection, hundreds of DNA are captured on the surface of silicon wafer or glass slide in large scale to detect hundreds of gene in one time. DNA chip, compared with traditional southernblotting and northernblotting, can detect a large amount of genes in one time and can be applied to detection of gene mutation, diagnosis of genetic gene and pharmagenetics. Currently, trend in this field is focused on developing method of low cost, rapidity, accuracy, simplicity and convenience.

One who go to hospital for a disease check usually feels so boring with complicated checking process and long waiting time for the result, if there is a portable diagnostic equipment that can make one get this kind of check at home or a first-aid room or in situ the check process will finish just in a minute. Such kind of check has been developed with the development of lab on a chip technique based on POCT in a large market scale. However, in situ check actually is limited much by requirement for certain instrument to confirm the checking result, so, development of a simple method for confirmation of checking result can greatly enlarge market scale so as to prepare for an aging and welfare society and pursue meaningful life.

Generally, the principle of DNA chip is to immobilize capture DNA with specific sequence using various immobilization method and find out which kind of capture DNA hybridize with target DNA or RNA. The hybridization between target DNA and capture DNA on chip is detected traditionally by radioisotope labels and expensive fluorescence dye scanned by microarray scanner.

Although the radioisotope label method is very sensitive and applicable to some fields, it is substituted by fluorescence dye probe for its environmental problem. Although fluorescence dye probe method is widely used for its advantage of high sensitivity, non toxicity, non destructivity and low cost, while in this method fluorescence dye molecules must be linked to biomolecules such as DNA and for signal readout the expensive laser or lamp scanner must be needed, so detection system using DNA chip is not widely used in commercial. Although mass spectroscopy method does not need biomolecule probe modification and provides structure information, it has the disadvantage of expensive and complicated instrument needed. Therefore, it becomes important to find a very simple method to detect DNA hybridization easily on DNA chip.

Hence, to develop a new detection method of DNA hybridization, the inventors for confirmation of target DNA hybridization have synthesized probe DNA immobilized nanoparticIe-DNA probe and reacted with target DNA, then detected hybridization degree of target DNA with capture DNA through detection of light scattering of nanoparticle probes on DNA chip. [Disclosure] [Technical Problem]

Object of the invention is to provide detection method of DNA hybridization on DNA chip through detection of light scattering of nanoparticle probes hybridized on DNA chip. [Technical Solution]

To achieve the above objection, we detect the light scattering of nanoparticle probes hybridized on DNA chip and get the information of DNA hybridization.

[Advantageous Effects]

As mentioned above, because the invention is to present new detection method of DNA hybridization on DNA chip, it can not only easily detect DNA hybridization on DNA chip using low cost lamp scanner without modification of biomolecules with fluorescent dye molecules as well as expensive optical instrument but also contribute to widely application of DNA chip. [Description of Drawings]

Figure 1 is to show general hybridization process used in detection of DNA hybridization between capture DNA and target DNA sample, target DNA and probe DNA on DNA chip (A) and modified hybridization process called sandwich hybridization (B) in which nanoparticle-DNA probe is used for detection of DNA hybridization according to light scattering of nanoparticle-DNA probes on DNA chip in the invention.

Figure 2 is synthesis of dye doped nanoparticle, which are modified further for preparation of probe nanoparticle DNA used in the invention, using water-in-oil microemulsion method (extra picture is TEM image of nanoparticle with diameter of about 50 run)

Figure 3 is the process of surface immobilization of dye-doped silica nanoparticles with probe DNA to make TMR-NP-DNA probe used for detection of DNA hybridization in the invention.

Figure 4 is the process of immobilization of oligonucleotide probe on glass slide through covalent bonding between sulfur and DNA to give capture DNA used for detection of DNA hybridization in the invention.

Figure 5 show hybridization of probe nanoparticle DNA with capture DNA directly which is different from Figure 1 (B) in which target DNA hybridized first with capture DNA, then probe nanoparticle DNA hybridize with other region of target DNA used for detection of DNA hybridization through light scattering of nanoparticle-DNA probes in the invention.

Figure 6 show format of HPV DNA chip used in the invention and laser scanning image of the result of sandwich hybridization with HPV16 target DNA using TMR-NP-DNA probe in the invention. Figure 7 is the laser scanner image and digital camera image of the result of sandwich hybridization with HPV16 target DNA using TMR-NP-DNA probe in the invention.

Figure 8 is DNA hybridization result shown as graph of S/N ratio (signal/noise) in scattering signal of both TMR-NP-DNA probes (one nanoparticle

3 4 contains about 10-10 dye molecules) hybridized on DNA chip and same concentration of TMR-DNA when hybridize on DNA chip (in case of TMR-NP-DNA probes, the ratio of (S/N)scattering to (S/N)fluorescence is between 0.7 and

-3

1.0, while, in case of only TMR dye, the ratio is below 10 ),

Figure 9 is the laser scanner image and digital camera image of the result of DNA hybridization using several kinds of probes. [Best Mode]

The invention is to provide a new detection method of DNA hybridization based on detection of light scattering of nanoparticle-DNA probes hybridized on DNA chip.

In detail the invention of detection method of DNA hybridization on DNA chip for disease checking includes reaction step of hybridization of probe nanoparticle DNA with capture DNA to form hybridized probes; step of removing not hybridized probe nanoparticle DNA after above reaction; step of detection of light scattering degree of hybridized nanoparticle-DNA probes.

In detail the invention of detection method of DNA hybridization on DNA chip for disease checking includes reaction step of hybridization of sample DNA with capture DNA to form hybridized complex; step of removing not hybridized sample DNA after reaction; reaction step of hybridizaton sample DNA on capture DNA chip with probe nanoparticle DNA; step of detection of light scattering degree of hybridized nanoparticle-DNA probes.

And in the invention of detection method of DNA hybridization on DNA chip, it is desirable to select among the group of silica, gold, polystyrene and quantum dots as nanomateria1s, here, silica nanomaterials are much more desirable.

And in the invention of detection method of DNA hybridization on DNA chip, it is possible to use radioisotope and fluorescent dye as fluorescent component of nanoparticle, here, TMRCtetramethyl rhodamine) are much more desirable.

And in the invention of detection method of DNA hybridization on DNA chip, it is desirable to use laser scanner and digital camera as detection instrument .

The following attached drawing is to explain more detail about the invention of detection method. What is mentioned below is the technique part of the invention and so the range of the invention is not limited to it.

Figure 1 General detection method (A) of DNA hybridization on DNA chip and sandwich hybridization process (B) using probe nanoparticl DNA with light scattering of hybridized probe nanoparticle DNA as detection method in the invention. And figure 1 (B) shows detection method based on light scattering to detect hybridization between target DNA and capture DNA on DNA chip. That is, the detection method of DNA hybridization of the invention is actually similar with general detection method based on complementary DNA hybridization to hybridize target DNA with capture DNA, target DNA with nanoparticle-DNA probe. Detection method of general DNA hybridization process is as following.

Capture DNA which hybridize with complementary target DNA is immobilized on surface of silicon wafer, glass slide or plastic slide (capture DNA used in the invention is also called probe DNA in some papers, while in the invention we call it capture DNA which immobilized on chip surface and hybridize with target DNA), target DNA with specific sequence hybridize with capture DNA and the rest region of target DNA hybridize with probe nanoparticle DNA. Not hybridized target DNA in sample and buffer solution are removed, we can know what kind of target DNA are present in sample through detection of signaling probes. Although not shown in figure, sometimes for PCR amplification reaction, fluorescent dye molecules have been introduced from the beginning of the reaction to conjugate fluorescent dye molecule with target DNA.

However, in our invention instead of using normal detection method on DNA chip which use DNA probes (figure 1 (A)Conjugated with fluorescent dye molecules, our detection method of DNA hybridization is based on detection of light scattering and nanoparticles are used as signaling probes with no need of fluorescent dye molecules for signaling. Rather than, it is important to control nanoparticle size and concentration to get certain degree of light scattering, and the conjugation methodCfigure 1 (B)) of nanoparticle-DNA is important.

Laser scanner for fluorescence detection is very expensive for its expensive detector and optical device, inconvenience in DNA chip becomes more obvious because laser scanner with considerable output power is needed for rapidity and accuracy in such detection system. Hence, in our invention instead of detection of fluorescence, light scattering is used in detection of DNA hybridization on DNA chip. Light scattering phenomenon used in detection of DNA hybridization is dependent on nanoparticle size and in proportion to six square of nanoparticle radius, the volume and mass of nanoparticle increase 1,000 times when the radius of nanoparticle increases 10 times, while light scattering of nanoparticle increase one million times.

Detection of DNA hybridization on DNA chip based on light scattering in our invention is as following.

Sample is injected into chamber of DNA chip which contain capture DNA. When there are Target DNA with specific sequence in the sample, Target DNA hybridize with complementary capture DNA and not hybridized part of target DNA are left. To detect such hybridization, in contrast to tradition, probe DNA conjugate with nanoparticle and we call it probe nanoparticle DNA. DNA probe conjugated with nanoparticle (probe nanoparticl DNA) hybridize with the remaining region of target DNA, such hybridization is called sandwich hybridization. Through such sandwich hybridization on DNA chip, we can detect hybridization of target DNA with capture DNA based on detection of light scattering of hybridized probe nanoparticle DNA.

Nanoparticle is not just the meaning of particle with nanometer size. Particles of several micrometer can be seen by naked eye or by digital camera, smaller particles can also be observed by low cost laser scanner (flat bed scanner) based on light scattering. That is, even if the nanopart icles are small enough we can also detect light scattering phenomenon of nanopart icles using low cost instrument.

Figure 2 show scheme of the other kind of nanoparticle used in our invention-dye-doped silica nanoparticle synthesis in water-in-oil microemulsion. And TEM image of such nanoparticles with diameter of 50 nm.

Figure 3 is the scheme of conjugation of DNA probe with silica nanoparticle to form probe nanoparticle DNA used in our invention of detection method of DNA hybridization. The synthesis of probe nanoparticle DNA will be described more detail in following part.

Firstly, silica nanoparticles are used and amine groups are introduced to the surface of silica nanoparticles. For example, amine groups are introduced to the surface of silica nanoparticles through silanization of 3- aminopropyltrimethoxysilane. Silanization is widely used method of introducing amine, sulfide, hydrocarbon groups to the surface of silica nanoparticles. The process is as follow, in reaction on glass slide surface, the R is substituted with OH group. RGl(OMe)₃ 3H₂O

Hydrolysis i- 3MeOH

R ^SSii((OOH)₃

2Si(OH)₃

2H₂O

Condensation

R R R I I

HQ Hi O - Si — O — Sl OH

I OH OH OH

OH OH OH

Substrate

Hydrogen bonding

Bond formation

Substrate

In addition, for introducing amine group to the surface of silica nanoparticle, the modification process using 3-aminopropyltrimethoxysilane is as follow.

3-aminopπ>py<frimethoxysi!afle xylene in vaccum

Hence, the silanized amine group can link to specific protein through interaction with aldehyde group. For example, through glutaraldehyde, avidin can be attached to the surface of silica nanoparticles. Such avidin attached silica nanoparticle can react with 3'-biotinalated DNA through specific avidin-biotin interaction to conjugate nanoparticle with DNA probe, thus probe nanoparticle DNA as mentioned above can be synthesized. Although it is obvious to us, detailed process is as follow.

Because both avidin and antibody are protein which have NH2 group on one end, the following method is used in all case. Here the immobilization of avidin is mentioned in detail.

GlutarBWeHytlfr

Avidin as well as streptavidin are widely used for their strong interaction with biotin. Avidin has four reaction region due to its tetramer structure. And biotin is one kind of vitamin H small molecule and is widely used to link to one end of DNA. The whole process is to react avidin attached nanoparticle with 3'-biotinalated DNA and wash repeatedly to give probe nanoparticle DNA. Currently, silica particles with different size can be ordered easily, amine-modified or carboxy-modified silica nanoparticles can also be synthesized or ordered easily.

There are many kinds of methods to conjugate DNA with nanoparticles. For example, the interaction between sulfur and DNA can be used. In figure 4, interaction between sulfur and DNA is used to prepare probe nanoparticle DNA which are used in our invention of detection method of DNA hybridization. Figure 4 shows the silanization of nanoaparticle and immobilization process of capture DNA. The above method is to introduce thiol group and immobilize oligonucleotide on glass slide to obtain capture DNA which hybridize with target DNA. It is possible to introduce amine, carboxy, sulfide and hydrocarbon group to the surface of nanoparticle through silanization to conjugate with DNA. Choosing what kind of conjugation method is dependent on individual, although what we show on the picture is just one kind of method, the technique stage of our invention is not limited on this. Such conjugation method of immobilization DNA on surface of nanopartilce based on strong interaction between avidin and biotin which is widely used and not limited to here, obviously, there are many kinds of conjugation method to prepare probe nanoparticle DNA.

On one side, in the range of our invention, besides conjugation of nanoparticle with probe DNA, conjugation of nanoparticle with target DNA is also possible, which is shown in figure 5. In detail, nanoparticle is conjugated with target DNA. Then, on DNA chip on which capture DNA is immobilized, the nanoparticle-target DNA hybridize with capture DNA. Finally, through detection of light scattering of nanoparticles hybridized on DNA chip, hybridization between target DNA and capture DNA can be detected. Target DNA-nanoparticle can be synthesized as mentioned before, for example, through interaction between avidin and biotin after introduction of biotin to target DNA and introduction of avidin to nanoparticle. However, as shown in figure 1 (B), various method can be used dependent on individual obviously. Such example clearly show that using target DNA-nanoparticle can also detect hybridization between target DNA and capture DNA based on detection of light scattering of nanoparticles.

Hybridization between nanoparticIe-DNA and capture DNA is detected based on light scattering of nanoparticles hybridized on DNA chip in our invention. The process is performed on commercial HPV(Human Papillomavirus) DNA chip. There are 22 types of HPV DNA probes immobilized. We have detected HPV 16 target DNA on HPV DNA chip of disease check using synthesized probe nanoparticle DNA in sandwich hybridization and signal detection is performed by laser scanner(figure 6).

In addition, on HPV DNA chip, detection of hybridization of nanoparticle- DNA using TMR-doped silica nanoparticles is performed and signal is obtained using both laser scanner and digital camera(figure 7). The result is that hybridization between capture DNA and target DNA of HPV 16 can be detected according to signal of TMR dye scanned by laser scanner, signal detection with digital camera are also possible. That is, light scattering of nanoparticles based detection method can be used for detection of DNA hybridization actually.

In graph of light scattering, the ratio of S/N (signal/noise) of both TMR-doped nanoparticle-DNA with diameter of 50 nm hybridized on HPV DNA chip and same concentration of TMR-DNA hybridized are detected. The result is that the light scattering of TMR-nanoparticles increased compared to that of TMR dye only, and the ratio of (S/N)scattering/(S/N)fluorescence is between 0.7 ~ 1.0 in

-3 case of using TMR-nanoparticle, while that is below 10 in case of using TMR dye only(figure 8). That is, the signal of light scattering of nanoparticle is obviously confirmed compared to noise, which is also confirmed in light scattering of plain silica nanoparticles.

DNA hybridization using different probes are also detected and the result image is taken by both digital camera and laser scanner(figure 9). The result is that DNA hybridization using cy5 probes is not detectable by digital camera but only detectable by laser scanner. In contrast, DNA hyridization using plain nanoparticle probes is not detectable by laser scanner but only detectable by digital camera based on light scattering of nanoparticles. Detection of DNA hybridization is dependent on particle size, nanoparticle concentration and quality of material .

In synthesis of probe nanoparticle DNA used in above detection of DNA hybridization, avidin is desirable to be used for conjugation of probe DNA, and g1utaraldehyde is desirable to be used for conjugation with avidin after introduction of amine group through silanization of 3- aminopropyltriraethoxysilane on surface of silica nanoparticle.

Our invention of detection of DNA hybridization is different from traditional one in which signaling molecules are used, while in our method no such signaling molecules used.

[Mode for Invention]

Following embodtment is to describe process of our invention in more detail .

But, following imbodiment is only one example and this invention is not limited to this imbodiment.

<Mode 1> Synthesis of nanopartides

The inventor have synthesized nanoparticle to further prepare probe nanoparticle DNA used in detection of DNA hybridization based on light scattering. Silica nanoparticles or dye-doped silica nanoparticles are synthesized and used in this invention.

<1-1> Synthesis of dye-doped silica nanoparticles

The inventor have synthesized dye-doped silica nanoparticles. The synthesis procedure is shown in figure 2. TMR(tetramethyl rhodamine) is used and dye-doped silica nanoparticles are prepared throug neutralization of silica monomer (TEOS), in detail, silica monomer (TEOS) is mixed with surfactant and stirred continuously. The result is that certain size of dye-doped nanoparticles are synthesized.

The result is that dye-doped silica nanoparticles are synthesized with certain size, it is not just conjugation of fluorescent dye molecules but the combination of fluorescent molecules and silica particles to give new nanoparticles.

<l-2> Synthesis of plain slilica nanoparticles

The inventor do not use the same synthesis method used in mode 1-1 to prepare plain silica nanoparticles, but ordered the plain silica particles(0.3 μm) from Bangs Laboratories, and call the particles plain silica nanoparticles.

<Mode 2> Synthesis of nanoparticle-DNA probes

Nanoparticle-DNA probe is synthesized using silica nanoparticles prepared in above Mode 1. Amine group is introduced to the surface of silica nanoparticles which are synthesized in above Mode 1-1 and 1-2 (dye-doped siica nanoparticles and plain silica nanoparticles) through silanization of 3- aminopropyltrimethoxysilane. Then, avidin is attached to the surface of silica nanoparticles through glParaldehyde. Then, avidin immobilized silica nanoparticles react with 3'-biotinalated DNA to conjugate probe DNA with nanoparticles and give probe nanoparticle DNA. (figure 4) Because the procedure is clear and obvious to those skilled in the art, so omitted here.

<Mode 3> Detection of DNA hybridization based on light scattering of nanopartic1e~DNA probes

The above (Mode 2) synthesized probe nanoparticle DNA are used in detection of DNA hybridization based on light scattering on DNA chip. Following is about DNA chip of disease check and signal confirmation in detail.

<3-l> DAN chip of disease check

The DNA chip used in the invention is from Biomedlab.Inc. Twenty-two types of Capture DNA (sequence 1: 5'H2N-TATGTGCTGCCATATCTACTTCAGAAACTACATA-3' ) are immobilized on surface of glass slide. HPV DNA is the major etiologica agent of cervical cancer and related to several malignancy tumor.

<3-2> Hybridization and sandwich hybridization of nanoparticle-DNA probes On DNA chip of disease check in Mode 3-1 target DNA (sequence 2: 5'- ATTGMTCMGπATGTAGTTTCTGAAGTAGATATGGCAGCACATA-3) in buffer is injected into chamber of DNA chip to hybridize with capture DNA, and not hybridized target DNA and buffer are removed. Then, probe nanoparticle DNA (sequence 3: 5'- ACTTGATTCAAT-3' ) prepared in process 2 hybridize with the remaining region of target DNA. The hybridization procedure is omitted.

<3-3> Confirmation of DNA hybridization on DNA chip based on light scattering

The result of the above (Mode 3-2) DNA hybridization on DNA chip is confirmed. The result is shown on figure 7 with HPV 16 type target DNA, the concept of the invention using probe nanoparticle DNA on HPV DNA chip is confirmed. Figure 8 shows that only the nanoparticle-DNA hybridized region give light scattering signal, so one can confirm that hybridization do not occur between target DNA (sequence 1) and capture DNA (sequence 2)in other region. One of detection methods of light scattering is to illuminate laser beam on DNA chip from angle of 45 degree, thus the reflection scattering is directed to front and such scattering can be detected and compared.

The usefulness of detection method of DNA hybridization based on light scattering of nanoparticles with laser scanner and digital camera as detection instrument is confirmed (figure 7). Hybridization between capture DNA and target DNA of HPV 16 type is confirmed by signal detection using laser scanner according to TMR dye doped in nanoparticles. The same result is obtained when using digital camera to take signal without laser scanner. That is, because light scattering increases one million times as diameter of nanoparticles increases 10 times, it is much more clear when changing the size of nanoparticles.

The ratio of S/N (signal/noise) in light scattering of both dye-doped silica nanoparticles and plain silica nanoparticles is mentioned in Figure 8. The signal of light scattering of nanoparticle is obviously confirmed compared to noise, such phenomenon is also found in light scattering of plain silica nanoparticles. The ratio of S/N (signal/noise) of both TMR-doped nanoparticle-

3 4

DNA with diameter of 50 nm (one nanoparticle contains 10 ~ 10 dye molecules) hybridized on HPV DNA chip and same concentration of TMR-DNA hybridized are detected. The result is that the light scattering of TMR-nanoparticles increased compared to that of TMR dye only, and the ratio of (S/N)scattering/(S/N)fluorescence is between 0.7 ~ 1.0 in case of using TMR-

-3 nanoparticle, while that is below 10 in case of using TMR dye only. That is, the signal of light scattering of nanoparticle is obviously confirmed compared to noise, which is also confirmed in light scattering of plain silica nanoparticles.

To further confirm the efficiency of the invention, the result image taken by both digital camera and laser scanner is shown in figure 9 when using cy5 probes, TMR-doped silica nanoparticle probes and plain silica nanoparticle probes. The result is that DNA hybridization using cy5 probes is not detectable by digital camera but only detectable by laser scanner. In contrast, DNA hyridization using plain nanoparticle probes is not detectable by laser scanner but only detectable by digital camera based on light scattering of nanoparticles. In case of using TMR-doped silica nanoparticles, DNA hybridization are detectable by both laser scanner and digital camera, thus it is no need to use high cost laser scanner. The left bottom part is the region where DNA conjugated nanoparticles probes can be seen. It means that capture DNA hybridized with complementary target DNA with certain sequence, the event can also be seen by naked eye and digital camera, and low cost laser scanner can be used to detect the signal dependent on size of nanoparticles.

Claims

[CLAIMS] [Claim 1]

Detection method of DNA hybridization on DNA chip based on detection of light scattering of probe nanoparticle DNA. [Claim 2]

The method of claim 1, wherein the detection method includes following step,

1) step of Hybridization between probe nanoparticle DNA and capture DNA on DNA chip of disease check;

2) step of removing probe nanoparticle DNA not hybridized after hybridization;

3) step of detection of light scattering degree of hybridized nanopart ic1e-DNA probes.

[Claim 3]

The method of claim 1, wherein the detection method includes following step,

1) step of hybridization between sample DNA and capture DNA on DNA chip of disease check;

2) step of removing sample DNA not hybridized after hybridization;

3) step of hybridization of probe nanoparticle DNA with sample DNA on DNA chip;

4) step of detection of light scattering degree of hybridized nanopartic1e-DNA probes.

[Claim 4]

The method of claim 1, wherein the above nanoparticle is selected among the group of silica, gold, polystyrene and quantum dots. [Claim 5]

The method of claim 4, wherein the above nanoparticle is silica nanomaterials. [Claim 6]

The method of claim 1, wherein the nanoparticle has radioisotopes and/or fluorescent materials inside and/or outside nanoparticle. [Claim 7]

The method of claim 6, wherein fluorescent material is TMR(tetramethyl rhodamine) dyes. [Claim 8]

The method of claim 1, wherein detection instrument is laser scanner or digital camera.