JP2004117350A - Luminescence nanochannel sensor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To aim at a hydrophobic field given by presence of surfactant in pores with nanometer size for developing a new sensor function. <P>SOLUTION: In a nanochannel body thin film in which an oxide layer includes surfactant micella, presence of a target substance in a specimen is detected according to luminescence intensity of the thin film associated with recognition of the target substance by a luminescence recognition agent inside the nanochannel. <P>COPYRIGHT: (C)2004,JPO

Description

The invention of this application relates to a light-emitting nanochannel sensor. More specifically, the invention of this application is a nanometer-sized thin film useful as a sensor for biochemical analysis, trace component analysis, etc. in a wide range of fields such as medicine, hygiene, industry, agriculture, and environmental evaluation. A new light-emitting nanochannel sensor using a pore (nanochannel) structure.

Conventionally, attention has been paid to nanometer-sized pores to produce such pore (mesoporous) substances. In these conventional studies, a substance having pores is formed by using the surfactant as a template by hydrolyzing the alkoxysilane compound in the presence of the surfactant. For example, conventional techniques include the preparation of a mesoporous substance on a mica substrate (Non-Patent Document 1), the preparation of a mesoporous thin film by evaporating a solvent (Non-Patent Document 2), the patterning of a mesoporous thin film, and the function of a silane coupling agent. (Non-Patent Document 3) and the like have been reported.
Hong Yang, et al., Nature, Vol. 379, 22 Feb. 1996, p. 703-705 Yun Feng Lu, et al., Nature, Vol. 389, 25 Sep. 1997, p. 364-368 Hongyou Fan, et al., Nature, Vol. 405, 4May. 2000, p.56-60

However, for example, despite the above-mentioned studies, although the technical development of a substance having nanometer-sized pores and its thin film as a functional material has been suggested for use as a pH sensor, etc., The fact is that little progress has been made. For example, although ultra-trace analysis using a nanometer-scale pore structure is expected to be realized, it has not been embodied yet.

One of the reasons for this is that, in the prior art, a surfactant is used as a template for forming pores. In some cases, attention is not paid to the hydrophobic field due to This hydrophobic field may receive more attention for the development of its function as an analytical sensor or the like.

Therefore, the invention of this application has been made in view of the circumstances described above, and it has been found that a substance having nanometer-sized pores has a hydrophobic property given by the presence of the surfactant used in the production process. Focusing on the field, it is an object to provide new technical means that can be applied to sensors as its function.

The invention of this application solves the above-mentioned problems. First, there is provided a nanochannel sensor having a thin film of a nanochannel body in which an oxide layer includes surfactant micelles. The present invention provides a luminescent nanochannel sensor characterized in that the presence of a target substance in a sample is detected based on the luminescence intensity of a thin film accompanying the recognition of the target substance by the luminescent recognition reagent.

The invention of this application also provides, secondly, a light-emitting nanochannel sensor characterized in that the oxide layer of the nanochannel body is mainly composed of silicon oxide. Mixing a reagent and a sample solution, extracting and capturing the target substance recognized by the reagent together with the luminescence-type recognition reagent in the nanochannel, and detecting the presence of the target substance in the sample solution by the luminescence intensity of the thin film Fourth, a luminescence-type recognition reagent is impregnated in the nanochannel in advance, and the target substance in the sample solution is detected by the luminescence intensity of the thin film associated with capture and recognition of the target substance in the sample solution. Provided is a luminescent nanochannel sensor characterized by detecting its presence.

According to the above invention of the present application, in a nanochannel thin film in which an oxide encapsulates a surfactant micelle, the emission intensity of the thin film accompanying the recognition of a target substance by a luminescence-type recognition reagent in a nanochannel can be used in a sample solution. The new development of the sensor function is realized by detecting the presence of the target substance and utilizing the hydrophobic field given by the presence of the surfactant in the nanometer-sized pore.

発 明 The invention of this application has the features as described above, and embodiments thereof will be described below.

What is most characteristic is that in the invention of this application, the structure of the nanochannel sensor is such that the oxide layer encapsulates the surfactant micelle and holds the inside of the nanochannel as a hydrophobic field. In addition, the detection of the target substance in the sample solution is performed based on the emission intensity of the nanochannel thin film accompanying the recognition of the target substance by the luminescence-type recognition reagent in the hydrophobic field. Such a unique structure and a thin film of a nanochannel body that enables the function thereof can be considered as, for example, the structure of FIG. 1 when schematically shown as a silica layer.

The nanochannel body is preferably prepared by first heating or drying an oxide-forming alkoxide compound as a raw material and a surfactant-containing acidic alcohol solution so that the oxide layer contains the surfactant micelles. Can be manufactured.

Generally, when the concentration of the raw material in the solution is relatively low, micelles are formed in the process of evaporation to dryness, and these serve as templates to form nanochannel bodies. On the other hand, when the raw material concentration is high, the raw material and the like are melted under high temperature and pressure, and a nanochannel body is formed in the process.

酸化 物 As the oxide-forming alkoxide compound in this case, various compounds may be used as long as they form an oxide layer of the nanochannel structure. For example, a silicon alkoxide compound is typically used to form a silicon oxide layer. In addition, alkoxides of various types such as titanium, zirconium, hafnium, tantalum, niobium, gallium, and rare earth elements are considered. can do.

各種 Various surfactants may be considered for use with these alkoxide compounds. For example, typical ones include quaternary ammonium salt type surfactants as ionic surfactants. Further, a sulfonic acid type is also included. It may be a polyether-type nonionic surfactant. However, one of the preferable ones is a cationic quaternary ammonium salt type.

The use ratio of the alkoxide compound and the surfactant differs depending on the types of the two and the like, and is not particularly limited. In general, the molar ratio of the surfactant to the alkoxide compound is 0.01 to 0. .5 can be used as a guide.

The alkoxide compound and the surfactant are mixed in an acidic aqueous solution and heated. The heating temperature at this time can be up to the reflux temperature. Hydrochloric acid, sulfuric acid, or an organic acid can be mixed to obtain acidic conditions. Further, it is preferable to coexist low-boiling alcohols such as ethanol, propanol and methanol in the aqueous solution.

(4) After heating, the nanochannel body in the invention of this application is generated. In this case, the heating solution may be spread on a solid substrate, or the solution may be heated on the solid substrate. In this way, a thin nanochannel body as schematically shown in FIG. 1 can be obtained. This can be called a thin film. Of course, various solid substrates may be used. A ceramic substrate such as mica / alumina or a glass substrate may be used, or a metal or organic polymer substrate may be used.

発 光 For example, the light-emitting nanochannel sensor of the invention of the present application is constituted by a nanochannel thin film in which an oxide layer contains surfactant micelles that can be produced by the above process. The form is roughly classified into the following extraction type and impregnation type. FIG. 2 schematically shows the outline.

In the extraction type, for example, a luminescence-type recognition reagent is dissolved in an aqueous sample solution, and is extracted into a nanochannel by hydrophobic interaction while forming a complex with the target substance, and the target substance is extracted based on the fluorescence intensity of the thin film. To detect. On the other hand, in the case of the impregnated type, a luminescent recognition reagent is introduced in advance from the aqueous solution into the nanochannel, and then the target substance in the sample aqueous solution is collected by the luminescent recognition reagent present in the channel, and the fluorescence of the membrane is measured. The target substance is detected based on the intensity. This impregnation type enables simultaneous detection of various types of chemical substances by arranging nanochannel thin films having different recognition reagents on the same substrate.

In any of the above cases, the luminescence-type recognition reagent may be of various types, such as those capable of forming a complex with the target substance, those capable of binding by reaction, and those capable of physical capture binding. It can be. In the hydrophobic field in the nanochannel, it is possible to use the luminescent recognition reagent even if it has various functional groups as its molecular structure. For these luminescent recognition reagents, the luminescent function may be enabled by various methods. In addition, these reagents may be not only low molecular weight compounds but also macromolecules such as DNAs, proteins, enzymes and the like and biological ones.

In the invention of this application, as a method for maintaining the hydrophobicity of the nanochannel body, when the nanochannel body is immersed in water or an aqueous solution, the surface activity contained in the nanochannel (pores) is reduced. Since part of the drug micelles elutes in water or an aqueous solution and the hydrophobicity in the nanochannel may decrease with time, the inner wall of the nanochannel is subjected to a hydrophobic treatment in advance, and the surfactant micelle and this inner wall are It is also effective to suppress the elution of surfactant micelles into water or an aqueous solution by increasing the hydrophobic interaction of.

に は In such a hydrophobizing treatment, a hydrophobizing agent can be used in consideration of the affinity with the nanochannel body. For example, when the nanochannel body is composed of silicon oxide, an appropriate silane coupling agent, more specifically, a silane coupling agent having a mercapto group is considered as a preferable one.

条件 The conditions for such a hydrophobic treatment may be appropriately selected experimentally. As a more preferable method, in the production of the nanochannel structure or the nanochannel thin film of the invention of the present application as described above, the hydrophobic treatment agent is added and contained together with a surfactant during the formation of the nanochannel body. Is considered.

The addition ratio of the alkoxide compound and the surfactant for forming the nanochannel body is, for example, a hydrophobizing agent such as a silane coupling agent in a molar ratio of 0.3 to 1.2 times the former, and It is considered that the molar ratio is about 3 to 20 times.

For example, as shown in FIG. 2, the emission intensity of the nanochannel thin film may be measured by measuring a change in emission intensity due to excitation light irradiation, or by using another emission mechanism and its detection method. It may be based on.

Therefore, examples will be shown below, and embodiments of the invention will be described in more detail. Of course, the invention is not limited by the following examples.

1. Preparation of nanochannel thin film
Using a surfactant molecular assembly (micelle) as a template, a silica-surfactant nanochannel thin film having a nanometer-sized pore (nanochannel) structure was prepared.
<Preparation of thin film preparation solution>
-The composition (molar ratio) of the solution was as follows.

TEOS: EtOH: H ₂ O: HCl: CTAB = 1: 8.8: 5.0: 0.004: 0.075
CTAB: Cetyltrimethylammonium bromide TEOS: Tetraethyl orthosilicate 1) 9.7 mL of EtOH, 12.3 mL of TEOS, and 1 mL of 2.78 × 10 ⁻³ MHC were mixed and refluxed at 60 ° C. for 90 minutes.

2) 18.4 mL of EtOH, 1.519 g of CTAB, and 4 mL of 5.48 × 10 ⁻² M HCl were added to the refluxed solution, followed by stirring for 30 minutes.
<Thin film production>
1) 350 μL of the thin film solution obtained by the above preparation was dropped on a washed and dried glass substrate,
2) Spin coating (spin-coat method) (4000 rpm, 30 sec).
<Drying of thin film>
After spin-coating, it was dried at room temperature for 1 hour.
<Alkali treatment> (Neutralization of HCl contained in thin film)
・ Alkaline buffer used (NH ₄ Cl-NH ₃ )
Mix 0.1M NH ₄ Cl and 0.1M NH ₃ aq (about pH 10)
1) The dried thin film was immersed in an alkaline buffer for 20 minutes.

2) Rinse while replacing the alkaline buffer with ultrapure water, and immerse in ultrapure water for 20 minutes.
2. Characterization of thin film <X-ray diffraction>
FIG. 3 shows the results of X-ray diffraction of the thin film obtained by the above process. A peak is observed at 2θ of 2.0, which indicates that a periodic structure on the order of nanometers was formed in the thin film. Assuming that the nanochannel has a honeycomb-like structure as shown in FIG. 1, the distance between adjacent channels is calculated to be 5.1 nm from the 2θ value. Assuming that the thickness of the silica wall is 1 nm, the pore diameter of the channel is estimated to be about 4 nm. Simultaneous measurement of X-ray diffraction and differential scanning calorimetry confirmed that surfactant molecules were present in the channel up to 300 ° C., and that there was no significant change in the micro-ordered structure.
<Thickness>
The film thickness obtained by ellipsometry and step measurement with an atomic force microscope was almost the same, and was about 390 nm. Next, the thin film preparation solution was diluted with ethanol to control the thin film. FIG. 4 is a graph in which the film thickness is plotted against the mole fraction of TEOS in the solution for forming a thin film. It became clear that the film thickness was almost proportional to the content of TEOS.
3. Detection of Aluminum Ion by Extraction Type A nanochannel thin film containing surfactant molecular aggregates (micelles) formed on a substrate according to the above process was treated with a glass substrate together with 20 μM of 8-quinolinol-5-sulfonic acid (Qs) in FIG. The samples were immersed in aluminum aqueous solutions having different concentrations for 20 minutes, air-dried, and then the emission spectrum and intensity were measured in the air. FIG. 6 shows the dependence of the emission spectrum on the aluminum concentration, and FIG. 7 shows a plot of the amplification factor of the emission intensity (1 when no aluminum ion is present) with respect to the aluminum ion concentration. It can be seen that the emission intensity increases with the aluminum ion concentration and increases to about 7 times around 30 μM. FIG. 8 shows the relative ratio of the emission intensity to the aluminum ion concentration up to 5 μM. It can be seen from this that the detection of aluminum ions of 1 μM or less is possible.

The above results indicate that Qs and Al in the sample solution form a complex and are trapped in micelles in the nanochannel as shown in FIG. 9, and the amount increases with the Al concentration. Is shown. The above results demonstrate that aluminum ions on the order of μM (approximately ppb), and even on the order of 1 μM or less, can be detected with extremely high sensitivity and simple.
4. Detection of Magnesium Ion by Extraction Type The same thin film substrate as described above was immersed in aqueous magnesium solutions having different concentrations containing predetermined Qs (1 μM and 10 μM) for 20 minutes, air-dried, and the fluorescence spectrum and intensity were measured in the air. The results are shown in FIG. It can be seen that in any of the Qs concentrations, the emission intensity increases with the Mg concentration in a range where the Mg concentration differs by three orders of magnitude. From this, it is understood that the sensor of the invention of this application has a very wide measurement concentration range and is a substance detection method with a wide dynamic range. In addition, the higher the Qs concentration, the better the amplification ratio of the emission intensity with respect to the Mg concentration.
5. Detection of magnesium ion by impregnation type A thin film substrate similar to the above was prepared and immersed in a 10 μM, 200 μM, 2 mM Qs aqueous solution for 20 minutes each. This controlled the amount of Qs impregnated in the nanochannel. These substrates were immersed in Mg aqueous solutions having different concentrations for 20 minutes, air-dried, and the emission spectrum and intensity were measured in the air. The result is shown in FIG. It can be seen that, regardless of the Qs treatment concentration, the amplification rate does not monotonically increase but has a maximum at a certain Mg concentration. Further, the Mg concentration giving the maximum increases both with the Qs treatment concentration. This result indicates that the optimum detection concentration range of the sensor for magnesium can be controlled by changing the Qs treatment concentration. This means that the optimum detection concentration of the sensor can be set according to the sample. In addition, by integrating nanochannel sensors having different optimum detection concentrations on the same substrate, even if the target substance concentration is completely unknown, it is possible to determine the concentration without requiring another preliminary measurement. . Furthermore, by disposing nanochannel thin films having different recognition reagents on the same substrate, it becomes possible to detect many kinds of chemical substances simultaneously.
6. Detection of potassium ion 5. As in the case of the above, the thin film of the impregnated sensor was immersed in an aqueous solution of a fluorescent molecule recognition reagent, and the recognition reagent was introduced into the nanochannel.

As a recognition reagent,
N- (9-Anthrylmethyl) monoaza-18crown-6
Was used.

Thereafter, the substrate was immersed in an aqueous solution of KCl and NaCl (adjusted to pH 7.6), and the luminescence response to potassium ions and sodium ions was measured. FIG. 12 shows the result.

結果 The results in FIG. 12 show that the fluorescence intensity increases depending on the concentration of potassium ions, and that they hardly respond to sodium ions. That is, it is understood that the sensor is excellent as a potassium ion selective sensor.

The effect of high-concentration sodium ions coexisting in the solution is extremely small. From the above, the sensor shown in this example can be used for analyzing biological samples such as blood and urine.
7. Sensor Array In the same manner as described above, a thin film forming solution was dropped on a glass substrate at a plurality of locations and air-dried to obtain an array structure in which a plurality of circular spot-shaped (about 3 mmφ) nanochannel thin films were arranged. Next, this was immersed in an aqueous solution of Qs as a luminescent molecule recognition reagent, and introduced into a nanochannel.

Using the obtained sensor array, aqueous solutions containing aluminum ions (Al ³⁺ ) having different concentrations were dropped on each of the spot-like nanochannel thin films, and light emission at circular spots was measured.

FIG. 13 shows the results of detection by a 1/3 color CCD camera detector at an excitation wavelength of 365 mm. FIG. 14 shows a fluorescent image of the sensor array.

From this result, by employing a sensor array configuration, a plurality of sample solutions can be measured instantaneously. For example, as shown in FIG. It can be seen that the measurement can be completed in a single measurement. And, because of the outstanding substance-collecting properties of the nanochannel, the high-concentration collection of the molecular recognition reagents and the like results in strong fluorescence, eliminating the need for expensive, large-scale detectors, and being compact and power-saving. A portable CCD camera, a CMOS camera, or a mobile phone equipped with a consumer camera can be used, and a highly mobile minute measuring device can be constructed.

Simultaneous detection of multiple chemical species is also possible by impregnating thin film spots on the same substrate with fluorescent biomolecule recognition reagents having different functions.

FIG. 15 shows an example. 5 is a photograph showing the result of simultaneous analysis of Al ³⁺ ions and K ⁺ ions. In the Al ³⁺ detection row, the fluorescence increases with respect to the Al ³⁺ solution, but does not respond to the K ⁺ solution. This confirms the selective detection of Al ³⁺ . On the other hand, in the K ⁺ detection line, the fluorescence increases with respect to the K ⁺ solution, but does not respond to the Al ³⁺ solution. The selective detection of K ⁺ is confirmed.

Then, for the solution in which Al ³⁺ and K ⁺ coexist, the luminescence of each corresponding detection site increases, which confirms simultaneous detection.

According to the invention of this application, as described in detail above, it is possible to focus on the hydrophobic field provided by the presence of a surfactant contained in a nanochannel having nanometer-sized pores and to enable new development of a function as a sensor. Can be.

It is the figure which showed this about the thin film of a nanochannel body typically. It is the figure which showed typically about the extraction type and the impregnation type sensor. It is the figure which illustrated the result of the X-ray diffraction about the nanochannel body thin film in an Example. FIG. 3 is a diagram illustrating a relationship between a TEOS content and a film thickness in an example. It is a figure showing the molecular structure of 8-quinotanol-5-sulfonic acid (Qs). FIG. 3 is a diagram illustrating an aluminum concentration dependency of an emission spectrum (thin film). It is the figure which illustrated the response to the aluminum ion of the light emitting type nanochannel sensor (extraction type). It is a figure showing relative ratio of luminescence intensity to aluminum ion concentration up to 5 μM. FIG. 3 is a diagram showing a mechanism of extraction of an aluminum-quinolinol complex by micelles in a nanochannel. It is the figure which illustrated the response to the magnesium ion of the light emitting type nanochannel sensor (extraction type). It is the figure which illustrated the response to the magnesium ion of the light emitting type nanochannel sensor (impregnation type). It is a figure showing a measurement result of luminescence response to potassium ion and sodium ion. FIG. 4 is a diagram showing the correspondence between the concentration of aluminum ions and the light emission intensity for light emission by a sensor array. FIG. 3 is a diagram showing a light emission image by a sensor array. It is the figure which showed the example of the luminescence image of two element (ion) simultaneous analysis by a sensor array.

Claims (4)

A nanochannel sensor with a nanochannel thin film in which the oxide layer contains surfactant micelles. A luminescent nanochannel sensor characterized by detecting the presence of a target substance. 2. The luminescent nanochannel sensor according to claim 1, wherein the oxide layer of the nanochannel body is mainly composed of silicon oxide. The luminescence-type recognition reagent and the sample solution are mixed, and the target substance recognized by the luminescence-type recognition reagent is extracted and captured in the nanochannel, and the presence of the target substance in the sample solution is detected by the luminescence intensity of the thin film. The luminescent nanochannel sensor according to claim 1 or 2, wherein: 3. The method according to claim 1, wherein the nanochannel is preliminarily impregnated with a luminescence-type recognition reagent, and the presence of the target substance in the sample solution is detected by the luminescence intensity of the thin film associated with capture and recognition of the target substance in the sample solution. Luminescence type nano channel sensor.