Abstract:
A biosensor chip with nano-structures. The biosensor chip includes a RF biosensor, comprising an isolated substrate; a ground plane; a filtering circuit; at least one cell detection area with nano-structures and a protection layer. The RF biosensor can detect the existence of the cancer cells, high frequency biological effects and the cells relationship between transfers by noninvasive method. The RF biosensor according to the invention can provide high accuracy and sensitivity in cancer cells detection.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims the benefit of Taiwan Patent Application No. 100136156, filed on Oct. 05, 2011, in the Taiwan Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference. 
       BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to a biosensor chip, and more particularly to a biosensor chip with nano-structures. 
         [0004]    2. Description of the Related Art 
         [0005]    According to current research and development, biosensor chips are usually divided into capillary electrophoresis chips and combination type chips. Two kinds of chips can provide a fast, accurate, high volume and automatic operating platform for sequencing analysis of deoxyribonucleic acid (DNA), screening of human&#39;s diseases, screening and development of new pharmaceuticals, medicine release and control, and food/environment inspection. For example, DNA detection chips have been widely applied in gene detection. Therefore, biosensor chips can complete a goal that is not achieved by conventional bio-detection. For instance, present cancer detection manner is usually detected when patients have certain symptoms or are on the danger lists. However, by utilizing technique of biosensor chips, doctors can be assisted to detect initially different kinds of cancers within a few minutes to further understand cancer genetic factors of patients so as to carry out prevention of the development of the disease. Technically, the chips produced by biomicro-electromachanical systems (BioMEMS) process have already shown promising development. However, BioMEMS have the shortcomings of high degree of difficulty in processing, high costs and short service life to be resolved. 
         [0006]    Taking cancer cells as an example, when cancer cells within a body are increased to reach 10.sup.7 cells (about a tumor with a size of 0.2 centimeter), cancer cells carries out metastasis through angiogenesis of induced vessels. When cancer cells develop and reach a size of 1 centimeter, only then the cells can be observed by instruments (general physical examination). Unfortunately, the growth of cancer cells at this stage cannot be controlled completely, and the growing of cancer cells can be found by two manners of medical imaging and biochemical examinations in the early state. 
         [0007]    During image examination, normal medical inspection includes X-ray photographs, ultrasonic or computer tomography that are difficult to find tumors with a size below 0.8 centimeter. Although tumors can be found by image examination, the found tumors are larger than 0.9 to 1.0 centimeter and may already have begun metastasis. Hence the foregoing tumors may not be effectively controlled or cured. During chemical examination, specificity combination between molecules is usually applied. For example, patients who have contracted acquired immune deficiency syndrome (AIDS) are usually inspected for the number of Helper T cells and Cytotoxin T Cells within the body. Both cells can be specifically expressed with two kinds of protein molecules CD4 and CD8. Therefore, a monoclonal antibody has affinity with CD4 or CD8 to accurately capture these cells. At this time, if the monoclonal antibody is labeled with fluorescent molecules, the number of Helper T cells and Cytotoxin T Cells can be determined according to intensity of fluorescent signals after measurement. Some cancers must reach certain sizes before the tumor label substance can be detected. Moreover, some cancers do not secrete tumor label substance, hence the existence of these cancer cells may not be sensitively screened with tumor labeling and at the same time, the kind of cancer cannot be confirmed. Thus, the sensitivity and specialty of the method need to be enhanced. 
         [0008]    Another biochemical examination manner “DR-70” is different from a normal tumor label. Its principle is to detect substances produced within the human body when reacting with cancer cells. When cancer cells start entering intercellular matrix from cancer in situ, human connective tissues would produce fibrinogen degradation products (FDP). If the FDPs exceed in a normal value, it represents that cancer in situ has become an invasive cancer. DR-70 can detect cancer cells smaller than 10.sup.6 cells and is currently a sensitive chemical examination. However, the foregoing analysis may be infected by histoplasma capsulatum, pneumonia, acute infection, autoimmune disease, external trauma (trauma days smaller than thirty days) and interfered with physiological states of hemolysis and pregnancy. Therefore, developing a non-invasive, high sensitivity, high specialty, real-time and inexpensive inspection tools is very important issue. 
         [0009]    In addition, after studying patents and master/doctor thesis in Taiwan&#39;s universities, the research that relates to biosensor chips in these universities, such as National Taiwan University, National Cheng Kung University, National Tsing Hua University, National Chiao Tung University, National Yang-Ming University, and National Central University has great outcome. Regardless of microelectrospray nozzle chips, cell counting chips and DNA replication chips suitable for detecting protection samples, it has rich research results. However, the biosensor chips developed by Taiwans&#39;s academic units do not take microwave filters as a basic structure yet and do not operate a nanometer structure having high frequency electromagnetic wave (larger than 10 GHz) as a tool for detecting and eliminating cancer cells. Therefore, the radio frequency biosensor chip having nanometer structures has great innovation and creativity. 
         [0010]    To overcome the foregoing shortcomings, a biosensor chip capable of providing non-invasion, high sensitivity, high specialty, real-time measurement is necessary to overcome the defects of prior arts. 
       SUMMARY OF THE INVENTION 
       [0011]    In view of the shortcomings of the prior art, the inventor(s) of the present invention based on years of experience in the related industry to conduct extensive researches and experiments, and finally developed a radio frequency biosensor chip as a principle objective. The radio frequency biosensor chip has nanometer structures and detects the existence of cancer cells within animal bodies, high frequency characteristics and deteriorate cancer cells in a non-invasive manner. 
         [0012]    To achieve the foregoing objective of the invention, a radio frequency biosensor having nanometer structures is provided and includes an isolated substrate, a ground plane, a filtering circuit, at least one cell detection area, a plurality of nanometer structures and a protection layer. The ground plane is deposited at a rear surface of the isolated substrate through a semiconductor process. The filtering circuit is deposited on the isolated substrate through the semiconductor process and has a first signal input/output port and a second signal input/output port. The cell detection area is arranged in the filtering circuit and has equivalent capacitance effect. The nanometer structure is arranged in the cell detection area. The protection layer is coated on the filtering circuit and defines an opening on the cell detection area, the first signal input/output port and the second signal input/output port. 
         [0013]    According to a radio frequency biosensor chip having nanometer structures of the invention, the filtering circuit is a conductor-backed coplanar waveguide line structure. 
         [0014]    According to a radio frequency biosensor chip having nanometer structures of the invention, an operating frequency of the filtering circuit is at 10 GHz. 
         [0015]    According to a radio frequency biosensor chip having nanometer structures of the invention, a material of the nanometer structure is gold. 
         [0016]    According to a radio frequency biosensor chip having nanometer structures of the invention, the nanometer structure is a column or a cone structure. 
         [0017]    According to a radio frequency biosensor chip having nanometer structures of the invention, a height of the nanometer structure is between 100 nm and 5000 nm, and its diameter is between 10 nm and 500 nm. 
         [0018]    According to a radio frequency biosensor chip having nanometer structures of the invention, an operating frequency of the filtering circuit is between 10 GHZ and 50 GHz. 
         [0019]    According to a radio frequency biosensor chip having nanometer structures of the invention, the cell detection area is an interdigitated electrode, a low characteristic impedance transmission line or a circular resonator. 
         [0020]    According to a radio frequency biosensor chip having nanometer structures of the invention, a material of the filtering circuit is gold (Au) or silver (Ag). 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0021]      FIG. 1   a  is a structural diagram of a radio frequency biosensor chip; 
           [0022]      FIG. 1   b  is a side view of a radio frequency biosensor chip; 
           [0023]      FIG. 2   a  is a lumped-element equivalent circuit diagram of a bandpass filter; 
           [0024]      FIG. 2   b  is a lumped-element equivalent circuit diagram of a band rejection filter; 
           [0025]      FIG. 3   a  is a schematic diagram of the radio frequency biosensor chip according to a preferred embodiment of the invention; 
           [0026]      FIG. 3   b  is an equivalent circuit diagram of the radio frequency biosensor chip; 
           [0027]      FIG. 4   a  is a schematic diagram of a plurality of nanometer structures; 
           [0028]      FIG. 4   b  is a side view of a plurality of nanometer structures; and 
           [0029]      FIG. 5  is a schematic diagram of a frequency response of the radio frequency biosensor chip. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0030]    The foregoing and other technical characteristics of the present invention will become apparent with the detailed description of the preferred embodiments and the illustration of the related drawings. 
         [0031]    The invention discloses a new type of radio frequency biosensor chip  100  by taking dual-mode bandpass filter as a base and collocates a semiconductor process to realize circuits, thereby detecting amount and size of human hepatoma cells (HepG2) and performing high frequency biological response analysis, such as equivalent resistance, equivalent inductance, equivalent conductance, equivalent capacitor, dielectric permittivity, loss tangent (tan δ), quality factor Q and characteristic impedance of HepG2, as an initial research and development basis. The cell detection area  130  of the radio frequency biosensor chip  100  has equivalent capacitor effect to achieve a simple design method capable of reducing process cost and enhancing the accuracy and sensitivity during measurement. 
         [0032]    With reference to  FIG. 1   a  and  FIG. 1   b  for a structure and side view diagrams of the radio frequency biosensor chip  100  is depicted. The radio frequency sensor chip  100  comprises an isolated substrate  150 , a ground plane  120 , a filtering circuit  110 , at least one cell detection area  130  and a protection layer  140 . The ground plane  120  is deposited at a rear surface of the isolated substrate  150  through a semiconductor process. The filtering circuit  110  is deposited on the isolated substrate  150  through a semiconductor process and has a first signal input/output port  111  and a second signal input/output port  112 . The cell detection area  130  is arranged in the filtering circuit  110  and has an equivalent capacitor effect. The protection layer  140  is coated on the filtering circuit  110 , and an opening is defined on the cell detection area  130 , the first signal input/output port  111  and the second signal input/output port  112 , wherein the filtering circuit  110  is a conductor-backed coplanar waveguide line structure. The isolated substrate  150  is a glass substrate. The material of the ground plane  120  is gold (Ag) or silver (Ag). The material of the filtering circuit  110  is gold (Ag) or silver (Ag). The cell detection area  130  is an interdigitated electrode, a low characteristic impedance transmission line or a circular resonator. According to a preferred embodiment of the invention, the materials of the filtering circuit  110  and the ground plane  120  are Au(2 μm)/Ni(20 nm). The operating frequency of the filtering circuit  110  is at 10 GHz, and the material of the protection layer  140  is a SU8 photo resistor. 
         [0033]    With reference to  FIG. 2   a  and  FIG. 2   b  for lumped element type equivalent circuits of a bandpass filter  200  and a band rejection filter  300  are depicted, wherein L.sub.1, L.sub.2 and L.sub.3 are inductors, and C.sub.1, C.sub.2 and C.sub.3 are capacitors and their input/output ends are represented with 50Ω impedance. In a first-order and plural-order filtering circuit  110 , the circuit composed of the interdigitated electrode, the low character impedance transmission line or the circular resonator have the equivalent capacitors (C.sub.1, C.sub.2 and C.sub.3). Cells can be placed in the equivalent capacitors that are taken as the cell detection area  130 . With reference to  FIG. 3   a  for a radio frequency biosensor chip  100  according to a preferred embodiment is depicted. The radio frequency biosensor chip  100  takes a dual-mode filter as a base. The length of the dual-mode filter is a full guided wavelength (λg). A ninety-degree electron length (a distance from a to b) is between an input end and an output end to have a perturbed structure (as the cell detection area  130  having equivalent capacitors). 135-degree electron length (a distance of a to c and b to c) is between the input/output ends and a perturbed point. Under a condition of no perturbed structure, the resonant frequency at the input end is activated, its output end may not generate resonance. However, two perpendicular modes generated by the circular resonator and the perturbed structure would reach coupling. The radio frequency biosensor chip  100  takes the conductor-backed coplanar waveguide line as a basis to have an advantage of conveniently designing the resonators and a semiconductor high frequency probe measurement with respect to ground-signal-ground (G-S-G)  111 ,  112 . A common dual-mode circular filter must have the following conditions: 
         [0034]    Ninety-degree electron length must be separated from the input end to the output end. 
         [0035]    A perturbed structure (taken as the cell detection area  130  having equivalent capacitors) is disposed in the circular resonator to allow the advancing of electromagnetic wave to have discontinuous phenomenon, thereby activating resonant frequency and generating bandpass. 
         [0036]    The circuit structure must be symmetrical. 
         [0037]    With reference to  FIG. 3   b  for the equivalent circuit of the radio frequency biosensor chip  100  is depicted. The dual-mode filter is an even mode and odd mode equivalent circuit. Zr is a characteristic impedance of the circular transmission line, and Zp is a characteristic impedance (since the characteristic impedance is lower, it can be an equivalent capacitor) of the perturbed structure, and Kz(Kz=Zp/Zr) is defined as ratio of two impedances. When the circular resonator is not attached with the perturbed structure Zp, its electron length is equivalent to a half-wavelength (a resonant frequency fr). The electron length of the perturbed structure Zp is 2θp. A position from d point to c point (the perturbed structure Zp) is θ.sub.1. A position from a point (or b point) to c point (the perturbed structure Zp) is θ.sub.2 (θ.sub.2=θ.sub.1−45 degrees). The resonant frequency f.sub.0e of the even mode and the resonant frequency f.sub.0d of the odd mode would satisfy: 
         [0038]    The resonant frequency f.sub.0e of the even mode: 
         [0000]        K   z  tan (θ 1   f′   0e )+tan (θ p   f′   0e )=0  (1)
 
         [0039]    f′.sub.0e=f.sub.0e/fr,f′.sub.0e is the resonant frequency of the even mode after normalization. The resonant frequency f.sub.0d of the odd mode is: 
         [0000]      tan (θ 1   f′   0o )+ K   z  tan (θ p   f′   0o )=0  (2)
 
         [0040]    f′.sub.0d=f.sub.0d/fr,f′.sub.0d is the resonant frequency of the odd mode after normalization. By regulating the electron length 2θp of the perturbed structure, the formation of passband can be immediately decided to form the frequency response for a classical dual-mode filter. The dual-mode filter has advantages of simple in design, small size and high passband attenuation to be suitable for a basic structure of the radio frequency biosensor chip. 
         [0041]    With reference to  FIG. 4  for a plurality of nanometer structures  131  are depicted. The nanometer structure  131  is a column or cone structure. In response to a preferred embodiment of the invention, the nanometer structure  131  is a column. Its material is gold, and its height (h) is between 100 nm and 5000 nm, and its diameter (D) is between 10 nm and 500 nm. In a process, the substrate  150  is directly delivered to a level furnace tube growing a silicon oxide (SiO.sub.2) layer in which a thickness is between 100 nm and 500 nm after rinsing with a standard RCA sequence, thereby preventing the substrate  150  from generating leakage current and conduction during measurement. Next, a test strip is rinsed through alcoholic solution. After drying with nitrogen gas, the test strip is placed into a vapor deposition system to perform vacuum pumping for 3 to 4 hours until a vacuum value reaches below 5×10.sup.−6 torr. A gold film then is deposited by vapor deposition. Its deposition condition is that: a thickness between 100 nm and 5000 nm; a temperature of the substrate  150  is below 200 degree Celsius through control. The highest evaporation rate is 1 angstrom/second. The test strip that is completed with film plating then undergoes a thermal oxidation process at different conditions via a vacuum annealing furnace tube, thereby growing gold nano-columns in which a temperature is about 400° C., 500° C. and 600° C.; a temperature retention time is about 1, 3 and 5 hours; and oxygen flow is about 10, 20 and 30 sccn. The Scanning Electron Microscope (SEM) is utilized to observe the material and electric properties of gold-nano columns, and an optimum condition of growing Titania nan-columns is obtained to provide a sample for subsequent analysis and surface plasma modification. 
         [0042]    In the measurement, its instrument needs to use a vector network analyzer (VNA) HP 8510C of high frequency probe stations  111 ,  112  containing G-S-G, wherein its measurement scope is between 0.045 and 50 GHz. With reference to  FIG. 5  for a frequency response of the radio frequency biosensor chip  100  is depicted. For example, cell amount in a culturing process can be obtained by a conventional cell counting method. The cells then are placed in the detection area of the radio frequency biosensor chip to observe the frequency response (including the variation of the center frequency, bandwidth, insertion loss and return loss under loaded cells and unloaded cells), thereby analyzing sizes, quantity and high frequency dielectric properties (e.g. the equivalent resistance (R) of hepatoma cells), inductance (L), conductance (G) and capacitance (C), dielectric coefficient and loss tangent) of cells. 
         [0043]    While using the network analyzer, error calibration must be carried out, including random errors, systematic errors, and drift errors. The random errors are mainly from noise of the system and the reliability of components in the instrument; these errors are unable to be eliminated by calibration. The systematic errors are mainly from the circuit design of the measurement instrument and can be eliminated by calibration. The drift errors are mainly that the performance of the system is different at different time and different conditions such as the change of ambient temperatures. This kind of errors can be eliminated by repeated calibration. The systematic error is reduced to below −50 dB after calibration, thereby decreasing the mistake during the measurement. After detecting the chip, the chip is immersed into phosphate buffer saline (PBS). An ultrasonic oscillator is utilized for 30 minutes to remove cells remained on the chip. After completing oscillation, the radio frequency biosensor chip  100  can be repeatedly used. In addition, the nanometer structure  131  can effectively pierce through cancer cells to transmit electromagnetic wave, in which the frequency is between 10 and 50 GHz, around the cancer cells to achieve the purpose of deteriorating the cancer cells. 
         [0044]    Hepatoma cells are taken as an example. When the nanometer structure  131  pierces through cells, the frequency response of the filter passband is shifted. According to the displacement quantity, the equivalent RLGC value, dielectric coefficient and loss tangent of cells can be further calculated. Four sets of Scattering parameters can be obtained from the displacement quantity, the parameters are S.sub.11, S.sub.12, S.sub.21 and S.sub.22 respectively, and these parameters are put into formula (3) to obtain a propagation constant γ(f): 
         [0000]    
       
         
           
             
               
                 
                   
                     
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         [0000]    and plural propagation constants can be rewritten into: 
         [0000]      γ( f )=α t ( f )+ j β( f )  (4)
 
         [0045]    wherein αt(f) is an attenuation constant, and β(f) is a phase constant, and both constants are: 
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         [0046]    with formula (6), an effective dielectric coefficient εeff(f) can be obtained. The characteristic impedance Z.sub.0 is: 
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         [0047]    According to the following formula, the equivalent resistance (R), the inductance (L), the conductance (G) and capacitance (C) of cells can be obtained: 
         [0000]      γ( f )× Z   0 ( f )= R+jωL   (8)
 
         [0000]      γ( f )/ Z   0 ( f )= G+jωC   (9)
 
         [0048]    With the following formula, the dielectric coefficient of the cells can be obtained: 
         [0000]      ε eff =(1 −q )+ qε   r   (10)
 
         [0049]    wherein q is a structure factor of the microstrip, and its loss tangent can be represented as the following: 
         [0000]    
       
         
           
             
               
                 
                   
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         [0050]    The invention improves over the prior art and complies with patent application requirements, and thus is duly filed for patent application. While the invention has been described by device of specific embodiments, numerous modifications and variations could be made thereto by those generally skilled in the art without departing from the scope and spirit of the invention set forth in the claims.