Patent Application
20080293148
The present invention relates to system and method for measuring a bio-element, and more particularly to system and method for measuring a bio-element capable of accurately detecting whether a bio-element such as protein, gene and the like is present in an atmosphere or vapor phase having controlled temperature and humidity thereof and measuring a content of the bio-element.
In recent years, there have been actively performed researches for developing a cantilever based sensor so as to detect a physical phenomenon or chemical reaction, which sensor is manufactured through a micro electro mechanical system (MEMS) process.
The cantilever based sensors that are currently researched adopt a manner of measuring a mass change with a light source such as laser, which change results from heat or gas adsorption in the atmosphere or liquid. In other words, most of cantilever based sensors uses an optic system to measure a static deflection due to a surface change of the cantilever.
An example of applying the sensing method by the deflection to detection of a bio-element is introduced in Nature Biotechnology 19, 856-860 (2001) and Science 288, 316-318 (2000), which discloses a method of detecting protein and gene through a biological reaction occurring on a surface of a micro cantilever. The sensing methods by the static deflection are carried out in a manner of illuminating a surface of the cantilever with a light source such as laser on and focusing the light with a position sensing diode to detect whether or not the protein or gene. However, the detection of the biological reaction in the system measuring a displacement of the cantilever is mainly performed in the liquid. In the optical measurement method of the cantilever in the liquid, there exist experimental errors such as signal attenuation, parasitic deflection, narrow dynamic range and the like due to the measurement in the liquid.
In the mean time, in addition to the optical measurement method for the cantilever displacement, there has been progressed a research on a micro cantilever sensor using a frequency change. Thundat et al. reported that it was possible to measure changes of spring constant by adsorption of Na+ ions on a surface of a micro cantilever sensor through a resonant frequency measurement (Applied Physics Letters 80, 2219-2221 (2002)). Also, the IBM research institute in Zurich, Swiss and some researchers reported that it was possible to detect specific gases in the air through the resonant frequency measurement. For example, U.S. Pat. No. 5,719,324 discloses a cantilever sensor using a reaction of chemical materials on a cantilever, wherein a variation of resonant frequency is used to analyze a target chemical material. In addition, U.S. Pat. Nos. 6,212,939 and 6,289,717 disclose a chemical sensor using adsorption on a silicon cantilever and a sensor combining a binding partner of a material which is desired to detect on a cantilever and then detecting the material.
However, since the sensors disclosed in the U.S. Patents detect the reaction in the liquid phase and the variations of the displacement or resonant frequency of the cantilever in the liquid using an optic method or detect the variation of resonant frequency through an exterior oscillator, they have the problems due to the measurement in the liquid as described above. In addition, the prior art is limited to a chemical material of vapor phase (U.S. Pat. No. 5,719,324) or focuses on the resonance using an external piezoelectric material or the sensing method using optics. Although U.S. Pat. No. 6,289,717 mentions a micro mechanical antibody sensor, it is limited to the measurement of the cantilever displacement by an optical method in the liquid, rather than an electric measurement method using a cantilever. Additionally, it is suggested a method of detecting prostate specific antigen (PSA; indicator protein of prostatic cancer) by a bending method in Nature Biotechnology, 19 pp 856-860. This suggestion is also a research on a liquid phase reaction of target molecules and an optical measurement method.
As described above, the cantilever sensors according to the prior art can measure the adsorption of liquid or vapor phase and biological reaction. However, the electric measurement in the liquid rather than optical measurement has large experimental errors due to the variations of resonant frequency as density and viscosity of the liquid are varied, and should endure low sensitivity due to damping. In addition, the measurement for antigen-antibody reaction using the prior cantilever sensor is mainly performed at room temperature or less (about 28° C.).
Accordingly, the present invention has been made to solve the above problems. An object of the invention is to provide system and method for measuring a bio-element capable of accurately detecting whether a bio-element such as protein, gene and the like is present in an atmosphere or vapor phase having controlled temperature and humidity thereof and measuring a content of the bio-element.
In order to achieve the above object, there is provided a bio-element measuring method comprising steps of: preparing a cantilever sensor having a plurality of cantilevers; measuring a basis resonant frequency for the plurality of cantilevers; reacting the cantilevers with a sample including a bio-element; measuring resonant frequencies of the cantilevers after the reaction, in a closed system that is isolated from an exterior environment and temperature and humidity thereof are controlled to a specific state; and calculating variations of the resonant frequencies of the cantilevers before and after the reaction to carry out a quantitative analysis of the bio-element included in the sample.
Preferably, the method of the invention may further comprising a step of cleaning and drying the cantilevers using one of ultra pure water and buffer solution before measuring the resonant frequencies of the cantilevers after the reaction with the sample.
Preferably, at least one of the cantilevers may have a molecule recognition layer on at least one of upper and lower surfaces of the cantilever.
Preferably, at least one of the cantilevers may don't have a molecule recognition layer on at least one of upper and lower surfaces of the cantilever.
Preferably, the molecule recognition layer may comprise a monoatomic layer.
Preferably, the temperature of the closed system may be 10˜60° C.
Preferably, the temperature of the closed system may be 30˜40° C.
Preferably, the humidity of the closed system may be a relative humidity of 10˜90%.
Preferably, the humidity of the closed system may be a relative humidity of 70˜90%.
Preferably, the plurality of cantilevers may be integrated with a piezoelectric film.
Preferably, the plurality of cantilevers may be integrated with a piezoresistive film.
According to another embodiment of the invention, there is provided a bio-element measuring method comprising steps of: preparing a cantilever sensor having a plurality of cantilevers; measuring a basis displacement value for the plurality of cantilevers using optic means; reacting the cantilevers with a sample including a bio-element; measuring displacement values of the cantilevers after the reaction, in a closed system that is isolated from an exterior environment and temperature and humidity thereof are controlled to a specific state; and calculating variations of displacement values of the cantilevers before and after the reaction to carry out a quantitative analysis of the bio-element included in the sample.
According to another aspect of the invention, there is provided a bio-element measuring system comprising: a closed system defining a predetermined space and isolated from an exterior environment; a sample supply system provided in the closed system and supplying and discharging a sample including a bio-element; a reaction chamber provided in the closed system, connected to the sample supply system to provide a space capable of receiving the sample and mounted with a cantilever sensor having a plurality of cantilevers; temperature control means for controlling a temperature of the closed system; and humidity control means for controlling humidity of the closed system.
Preferably, at least one of the cantilevers may have a molecule recognition layer on at least one of upper and lower surfaces of the cantilever.
Preferably, at least one of the cantilevers may don't have a molecule recognition layer on at least one of upper and lower surfaces of the cantilever.
Preferably, the molecule recognition layer may comprise a monoatomic layer.
Preferably, the temperature in the closed system may be controlled in a temperature range of 10˜60° C., more preferably 30˜40° C.
Preferably, the humidity of the closed system may be controlled in a relative humidity range of 10˜90%, more preferably 70˜90%.
Preferably, the plurality of cantilevers may be integrated with a piezoelectric film.
Preferably, the bio-element measuring system may further comprise a power supply section and resonant frequency measuring means for measuring the resonant frequencies for the cantilevers.
Preferably, the bio-element measuring system may further comprise optic means for measuring displacement values for the cantilevers.
Preferably, the humidity control means may comprise storage means having a volume in which liquid for controlling humidity is stored.
Preferably, the liquid for controlling humidity may be one of ultra pure water and buffer solution.
Preferably, the sample supply system may comprise a sample inlet through which the sample flows in, a sample pipe connected to the reaction chamber and supplying the sample to the reaction chamber, and a sample outlet provided to a side of the reaction chamber and discharging the sample in the reaction chamber.
According to the invention, it is measured the basis resonant frequency or basis displacement value for the cantilevers before the cantilevers react with the sample including the bio-element to be measured. After the reaction with the sample, it is measured the resonant frequencies or displacement values in the closed system having temperature and humidity controlled to the specific state. Accordingly, it is possible to carry out a quantitative analysis of the bio-element in the sample through variations of the resonant frequencies before and after the reaction. Since the system and method of the invention adopt a measurement manner in the atmosphere or air, rather than in the liquid as the prior art, it is possible to eliminate the problems resulting from the measurement in the liquid and thus to perform accurate quantitative and qualitative analyses.
System and method for measuring bio-element according to the invention have following effects. According to the invention, it is measured the basis resonant frequency or basis displacement for the cantilever before the reaction with the sample having the bio-element to be measured included therein. After the reaction, it is measured resonant frequency or displacement for the cantilever in the closed system having temperature and humidity controlled to specific states. Through the measures, it is possible to perform the quantitative analysis of the bio-element included in the sample from the variations of the resonant frequency or displacement before and after the reaction.
In particular, according to the invention, since the measurements are carried out in the atmosphere or air, rather than in the liquid as the prior art, it is possible to solve the problems due to the measurements in the liquid, for example, the experimental errors such as the variations of the resonant frequency by the ostensible reasons such as viscosity or density of the liquid.
In addition, according to the invention, it is possible to solve the experimental errors and the sensitivity deterioration due to the parasitic deflection and narrow dynamic range which are problems of the displacement measurement method using the optic system according to the prior art.
Hereinafter, a preferred embodiment of the present invention will be described with reference to the accompanying drawings. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear.
A core of the technical spirit of system and method of measuring a bio-element according to the invention is that the bio-element is measured in the atmosphere or vapor state in a space having controlled temperature and humidity. It should be noted that
Referring to
The reaction chamber 101 has a predetermined reaction space for receiving the sample and a cantilever sensor is mounted in the reaction chamber 101. The cantilever sensor comprises a substrate 110 and at least one cantilever 120. The cantilever 120 is meant that it comprises a cantilever having a minimum structure. That is, the cantilever 120 applied to the invention comprises all cantilevers having a molecule recognition layer serving to trap a bio-element. For example, it is comprised all cantilevers having a molecule recognition layer which is a minimum structure condition as means for detecting a bio-element, such as cantilever having a separate piezoelectric transducer in U.S. Pat. No. 5,719,324 and cantilever having integrated a piezoelectric film or piezoresistive film in Korean Patent Application No. 2003-92618 that was filed by the applicant. The molecule recognition layer is formed on at least one of upper and lower surfaces of the cantilever 120 and comprises a detection film 126 made of a conductive material such as gold (Au) and a monoatomic layer 127 formed on the detection film 126, as shown in
The cantilever sensor shown in
In addition to the sample supply system 131, 132, 133 and the reaction chamber 101, the bio-element measuring system of the invention further comprises a cap 180 having a predetermined shape and isolating the sample supply system and the reaction chamber from the exterior environment. The bio-element measuring system 100 of the invention forms a closed system due to the cap 180. Meanwhile, the bio-element measuring system 100 comprises humidity control means and temperature control means 150. The humidity control means is means for controlling humidity of the closed system in the cap 180 and comprises storage means 141 at a side in the cap in which liquid for controlling humidity, for example ultra pure water (deionized water) or phosphate buffered solution (PBS) can be stored. An amount of the liquid for controlling humidity stored in the storage means 141 can be properly regulated depending on desired humidity. The temperature control means 150 serves to control a temperature of the closed system defined by the cap.
In the mean time, the cantilever sensor provided in the reaction chamber 101 may be further provided with a power supply section 170 supplying power to the cantilever 120 and means 160 for measuring a resonant frequency delivered from the cantilever 120. In addition, in case of measuring a displacement of a static deflection of the cantilever sensor, although it is not shown, an optic system comprising a laser diode for measuring the displacement may be provided to the bio-element measuring system.
Hereinafter, a bio-element measuring method according to the invention will be specifically described with reference to
First, according to the bio-element measuring method of the invention, a cantilever sensor is prepared (S601). The cantilever sensor is meant by a cantilever sensor comprising a molecule recognition layer that is the minimum structure element of the cantilever sensor for trapping a bio-element and includes all cantilever sensors having the molecule recognition layer. The molecule recognition layer comprises a detection film made of a conductive material and a monoatomic layer formed on the detection film and is sometimes meant by the detection film only or the monoatomic layer only, as described above. In addition, the cantilever sensor comprises a substrate and a plurality of cantilevers. At least one of the cantilevers may not be provided with the molecule recognition layer. A shape of the cantilever can be modified into a variety of forms depending on uses thereof. The cantilever shown in
When the cantilever sensor is prepared, an electric field is applied to the cantilever sensor to measure a basis resonant frequency of the cantilever (S602). At this time, the basis resonant frequency is measured to compare it with a resonant frequency after a reaction.
In the mean time, at least one of the cantilevers is subject to the resonant frequency measurement, under state that the molecule recognition layer is not provided to it. The reason to measure the resonant frequency for the cantilever having no molecule recognition layer is as follows. The essential characteristics of the bio-element measuring method according to the invention is to measure a basis resonant frequency for a cantilever having a molecule recognition layer and again to measure a resonant frequency for the corresponding cantilever after a reaction, thereby recognizing a bio-element through a variation of the resonant frequencies before and after the reaction. However, since a target sample also includes other bio-elements besides the bio-element to be measured, the other bio-elements can be trapped to the molecule recognition layer after the reaction. In this case, with regard to whether or not the correct bio-element and a content of the element, there may occur an error. In order to prevent the problem, a cantilever having no molecule recognition layer is provided. By providing the cantilever having no molecule recognition layer, it is possible to prevent a non-specific binding and to minimize a room for an error occurrence from a measurement environment.
When the bio-element measuring system shown in
In the mean time, as described above, the method of measuring a basis resonant frequency has been suggested to set a basis value before the reaction. However, it can be applied a method of measuring a displacement by static deflection of a cantilever as the basis value, in addition to the basis resonant frequency. In other words, displacements of a cantilever before and after the reaction are optically measured through a laser diode and the like to determine whether or not a bio-element trapped in the corresponding cantilever, and a content of the bio-element. At this time, the process of measuring the displacement before the reaction can be referred to as a process of measuring a basis displacement (S602). In addition, similarly to the measurement of the basis resonant frequency, the process of measuring a basis displacement is carried out under state that at least one of the cantilevers is not provided with the molecule recognition layer. The reason not to provide the molecule recognition layer to the cantilever is same as the case of the process of measuring the basis resonant frequency.
When the process of measuring the basis resonant frequency or basis displacement is completed, a reaction process proceeds (S603). The reaction process is meant that a sample including a bio-element to be measured, for example blood is reacted with the cantilever sensor. An example of the reaction process will be described with reference to the bio-element measuring system shown in
The sample such as blood is poured into the sample inlet 131 and flows in the reaction chamber 101 having the cantilever sensor via the sample pipe 132. Under state that the sample has flowed in the reaction chamber 101, a reaction is progressed for about 5˜100 minutes. Through the reaction, a bio-element to be measured, for example prostate specific antigen (PSA) which is an indicator protein of prostatic cancer is trapped on a surface of the molecule recognition layer, specifically monoatomic layer 127 (in
Under state that the reaction has been completed, the cantilever sensor is cleaned and dried (S604). The cleaning process may be carried out using a buffer solution such as phosphate buffered solution (PBS). When the bio-element measuring system shown in
Under state that the washing and drying processes have been completed, a process of measuring a bio-element is progressed in earnest. The process of measuring a bio-element is performed in the closed system having controlled temperature and humidity thereof to a specific state (S605, S606). The closed system is meant that the cantilever sensor having completed the reaction is isolated from an exterior environment. The bio-element measuring system shown in
First, the reason to fix the temperature and humidity in the closed system to a specific state is to optimize the determination of whether or not the bio-element trapped in the cantilever sensor and a content of the bio-element. For doing so, the temperature and humidity should be able to be controlled. The temperature control means 150 and the humidity control means serve to control the temperature and humidity, respectively. The temperature control means may comprise a heating block and a control device thereof. The humidity control means is provided with the storage means 141 in which the liquid for controlling humidity, for example ultra pure water (deionized water) or PBS is stored to properly control the humidity in the closed system, as described above with regard to
In order to find an optimal measurement condition, and the content of the bio-element, the temperatures and humidity were variously applied in experiments relating to the invention. Specifically, it was checked behaviors of the resonant frequency change as the temperature was varied under fixed humidity conditions and as the humidity was varied under fixed temperature conditions. The specific conditions of the temperature and humidity were 10˜60° C. and 10˜90% relative humidity.
As shown in
In
In the mean time, in the graph (B) of
Under state that the optimal temperature condition for the cantilever measurement is determined from the graph in
In the mean time, the result as shown in
From
Through a series of processes of preparing the cantilevers, measuring the basis resonant frequency, carrying out the reaction, measuring resonant frequencies after the reaction, calculating the variations of resonant frequency before and after the reaction and the like, it is possible to perform a quantitative analysis of the bio-element in the sample. In addition, since it is possible to determine the reaction degree, i.e., behaviors of the bio-element from the variations of relative humidity or temperature under specific temperature or relative humidity conditions, characteristics of the bio-element can be easily determined.
In the mean time, the invention has been described on the basis of the resonant frequency as shown in
In addition, although the embodiments of the invention have been described with reference to the indicator protein of prostatic cancer (PSA) as the bio-element, it is possible to detect a variety of bio-elements such as DNA, cell and the like, in addition to the PSA. Additionally, although the embodiments have been described on the basis of the variations of resonant frequency or displacement using the cantilever, it is possible to make progress the above described processes (S601˜S606) by applying a quartz crystal mass balance (QCM) used for the prior art, instead of the cantilever.
System and method for measuring bio-element according to the invention have following effects. According to the invention, it is measured the basis resonant frequency or basis displacement for the cantilever before the reaction with the sample having the bio-element to be measured included therein. After the reaction, it is measured resonant frequency or displacement for the cantilever in the closed system having temperature and humidity controlled to specific states. Through the measures, it is possible to perform the quantitative analysis of the bio-element included in the sample from the variations of the resonant frequency or displacement before and after the reaction.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2004-0074116 | Sep 2004 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR05/03066 | 9/15/2005 | WO | 00 | 3/14/2007 |
