Abstract:
A method of detecting a biological analyte within a sample ( 12 ) is provided. The analyte is one that can be electrically charged or polarized in the presence of an electric field. The sample is place in proximity with a ferroelectric transducer ( 14 ). An electric field is established to polarize the analyte in the sample. An electric response of the ferroelectric transducer resulting from the electric field and indicative of the presence of the analyte in the sample is then sensed. Also provided is a sensor for detecting the analyte within the sample. The sensor has a ferroelectric transducer and first ( 20 ) and second electrodes ( 22 ) for establishing a potential difference across a sample disposed adjacent to the transducer to generate an electric field in the sample. The sensor may also have an electric signal detector ( 26 ) for sensing the electric response.

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]     This application claims priority from U.S. provisional application no. 60/540,069, entitled “FERROELECTRIC FILMS FOR BIOLOGICAL SENSING AND DETECTION APPLICATIONS” and filed Jan. 30, 2004, which is incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention relates generally to sensing analytes, and more particularly to method and apparatus for sensing biological analytes.  
       BACKGROUND OF THE INVENTION  
       [0003]     Biosensors are sensors for sensing biological analytes. Biosensors have wide-spread applications in various fields such as medicine, environmental protection, food processing, security, defence, and the like.  
         [0004]     Known biosensors can be classified based on their transduction methods, which include three main types—optical transduction, electrochemical transduction, and piezoelectric transduction. However, each of these three types of biosensors has some shortcomings.  
         [0005]     For example, optical biosensors may require delicate and expensive instrumentation. Low signal to noise ratios can result from ambient light. The dynamic range of detection can be small in comparison with electrical sensors. Further, signal intensity is dependent on sample volume and thus it may be difficult to detect a small volume of sample.  
         [0006]     The electrochemical biosensors typically have low sensitivity.  
         [0007]     The piezoelectric transducers in piezoelectric biosensors can be fragile which limits their application.  
         [0008]     Thus, there is a need for a biosensor or a transducer for biosensors that is relatively simple in structure, easy and inexpensive to manufacture, and/or highly sensitive. There is also a need for biosensors which has a disposable transducer.  
       SUMMARY OF THE INVENTION  
       [0009]     In one aspect of the present invention, there is provided a method of detecting a biological analyte within a sample. The analyte can be electrically charged or polarized in the presence of an electric field. The sample is placed in proximity with a ferroelectric transducer. An electric field is established to polarize the analyte in the sample. An electric response of the ferroelectric transducer resulting from the electric field and indicative of the presence of the analyte in the sample is sensed.  
         [0010]     In another aspect of the invention, there is provided a sensor for detecting a biological analyte within a sample, wherein the analyte can be electrically charged or polarized in an electric field. The sensor comprises a ferroelectric transducer; a biological sample disposed adjacent the transducer; first and second electrodes for establishing a potential difference across the sample to generate an electric field in the sample; and an electric signal detector for sensing an electric response of the ferroelectric transducer resulting from polarization of the analyte, and indicative of the presence of the analyte in the sample.  
         [0011]     Other aspects, features, and benefits of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]     In the figures, which illustrate exemplary embodiments of the invention,  
         [0013]      FIG. 1  is a schematic diagram of a biosensor;  
         [0014]      FIG. 2  is a cross-sectional view of a ferroelectric transducer;  
         [0015]      FIG. 3  is a schematic cross-sectional view of analytes immobilized on a transducer;  
         [0016]      FIG. 4  is a line graph of detected voltage/current versus concentration for a sample; and  
         [0017]     FIGS.  5  to  10  are line graphs of voltage shift versus concentrations for several biological samples. 
     
    
     DETAILED DESCRIPTION  
       [0018]      FIG. 1  is a schematic diagram of a biosensor  10  for detecting a biological analyte within a sample, exemplary of an embodiment of the present invention. A biosensor is a sensor suitable for detecting or sensing biological analytes. Biological analytes include proteins, DNAs, viruses, antigen-antibody, bacteria, fungus, drugs, and the like. Biosensor  10  is suitable for detecting biological analytes which can be electrically polarized or charged in the presence of an electric field. A biological sample, or biosample such as biosample  12 , is a sample that potentially includes one or more biological analytes.  
         [0019]     Biosensor  10  includes a ferroelectric transducer  14 . Transducer  14  may be generally plate-shaped or film-shaped and has a top surface  16  and a bottom surface  18 . The top surface  16  contacts biosample  12 . Biosensor  10  also includes two electrodes  20  and  22  for establishing a potential difference across biosample  12  and transducer  14 . Top electrode  20  is in contact with biosample  12  and bottom electrode  22  is in contact with bottom surface  18  of transducer  14 . Electrodes  20  and  22  are connected to source  24  that applies a voltage across electrodes  20  and  22  and hence biosample  12  and transducer  14 . In the depicted embodiment, electrodes  20  and  22  are flat plates.  
         [0020]     An electric signal detector such as voltmeter  26  or ammeter  28  is operably connected or positioned to detect an electric signal from the electrodes  20  and  22  when the voltage is applied.  
         [0021]     Transducer  14  is formed, at least in part, of a ferroelectric material such as Ba x Sr 1-x TiO 3  (BST) or Pb(Zr x Ti 1-x )O 3  (PZT), where “x” can be any number between 0 and 1. As will be appreciated, BST can become non-ferroelectric at temperatures above its Curie temperature, which is dependent on the value of “x”. The ferroelectric material may also be a ferroelectric polymer, which may or may not be doped with a doping element such as lanthanum or manganese. The ferroelectric material can be in an amorphous, polycrystalline, or nano-structured phase. The ferroelectric material may have any suitable shape and size. For example, it can form a thin film with a suitable thickness. Typical thickness can vary between about 160-200 nm, but can be up to, for example, 1 μm. A thicker film can allow a higher voltage thus increasing the sensitivity of the sensor, but may be more expensive and difficult to fabricate. Thus, it may not be economically desirable to have a ferroelectric layer thicker than necessary. A BST film about 180 nm thick has been found adequate for detecting certain biological analytes. Top surface  16  of transducer  14  may or may not be formed with a ferroelectric material. Top surface  16  may be formed or treated with a material suitable for immobilizing biosample  12  thereon. For example, top surface  16  may have a coating material to which target analytes can directly attach. Top surface  16  may also be coated with molecules that have specific affinity to the target analytes (referred to as “probe molecules”). Because probe molecules have specific affinity to the target analytes, they will selectively capture or bind the target analytes. Suitable probe molecules will depend on the target analyte and ferroelectric material used, as will be understood by persons skilled in the art.  
         [0022]     Electrodes  20  and  22  can be any suitable electrodes. Similarly, source  24  and signal detectors  26  and  28  can be any suitable source or detectors. Suitable electrodes, signal sources and detectors will be known to persons skilled in the art. For example, a multimeter or an oscilloscope may be used to measure both voltage and current from electrodes  20  and  22 . The signal source can be a direct current (DC) or alternative current (AC) source and may provide a constant voltage or current. As can be understood, source  24  and detectors  26  and  28  can be integrated. Top electrode  22  may be spaced from transducer  14  at a fixed distance, or may be moveable relative to transducer  14 .  
         [0023]     Transducer  14  and bottom electrode  22  can be formed on a silicon wafer using known semiconductor techniques. An exemplary procedure for forming a BST transducer on a silicon wafer is described with reference to  FIG. 2  for illustration purposes. As illustrated, a silicon wafer  30  is coated with a platinum layer  32 . A BST film  34  is formed on platinum layer  32 . As can be appreciated, platinum layer  32  is the bottom electrode and film  34  is the transducer. Film  34  can be formed by spin-coating a BST sol solution onto layer  32 . The BST sol solution can be prepared in any suitable manner, which can be readily determined or understood by a person skilled in the art.  
         [0024]     For example, BST sol solution can be formed by mixing commercially available titanium butoxide (Ti(OC 4 H 9 ) 4 ), barium acetate (Ba(CH 3 COO) 2 ) and strontium acetate (Sr(CH 3 COO) 2 ) to form a precursor solution, and adding acetylacetone (C 5 H 7 OOH) and 2-ethoxy-ethanol (C 4 H 9 OOH) to stabilize the solution. The BST sol solution can be spun onto the cleaned platinum layer  32  at 4000 rpm for 30 seconds. Multiple layers of BST can be coated to obtain a desired film thickness. For example, four layers of spin-coated BST can form a total thickness of about 180 nm. After final coating, the ensemble can be annealed, for example, at 475° C. in air for about one hour in a quartz furnace. In order to make electric connection to bottom electrode  32 , a portion of the BST film can be etched off, for example by using 1:5 di-ionized water diluted HF solution, so as to expose a portion such as portion  36  of bottom electrode  32 .  
         [0025]     Biosample  12  may be a liquid and can be introduced onto top surface  16  either by direct liquid dropping, such as by using a pipette (not shown), or through a fluid channel (not shown). The fluid channel may be small in cross-section which can be in the micrometer scale. Sensor  10  may be housed in a chamber which is in fluid communication with the fluid channel. For example, such a chamber and channel may be formed on a micro-chip. The biological analytes to be detected should be electrically polarisable or become charged under an electric field. The analytes in biosample  12  may be mobile or immobilized on top surface  16  ( FIG. 1 ) of transducer  14 . For example, analytes may be immobilized directly on top surface  16 . Alternatively, as shown in  FIG. 3 , the analytes in biosample  12 , such as analyte  38 , may be “captured” by or bond to probe molecules, such as probe molecules  40  which are attached to the top surface  16  of transducer  14 .  
         [0026]     The signal detector can detect one or more of voltage, current, electrical charge, resistance, capacitance or other electrical properties that can be different between signals obtained for biosample  12  and a reference sample. The signal detector can also include a circuit for signal amplification, noise deduction or other purposes.  
         [0027]     As can be understood, biosensor  10  can be a capacitive, resistive, diode or transistor type sensor. For example, biosensor  10  and biosample  12  together can form two parallel capacitors.  
         [0028]     In operation, source  24  establishes a potential difference (voltage) across electrodes  20  and  22  and hence biosample  12 . The potential difference may have a pre-selected voltage value or be adjusted to maintain a pre-selected current flow through electrodes  20  and  22 . The pre-selected voltage or current may vary depending on a number of factors such as the intended application, the transducer material and thickness, the analyte or sample type, distance between the electrodes, and the like. Typically, the voltage may be in the range of about 1 to 100 volts, and the current may be on the order of nA or μA.  
         [0029]     For ease of description, it is assumed below that a constant current (I) is maintained across electrodes  20  and  22 . The pre-selected current level can be maintained by monitoring the current through ammeter  28  and adjusting the output of source  24 , either manually or automatically. In any event, the potential difference establishes an electric field within biosample  12 . This field, in turn, polarizes or charges analyte (or a fraction thereof) within the biosample  12 .  
         [0030]     Biosample  12  is in proximity with transducer  14 . The target analyte in biosample  12  may be immobilized on top surface  16  of transducer  14 , either directly attaching to top surface  16  or by binding to probe molecules, such as probe molecules  40 , attached to top surface  16 . When an immobilization step is performed, the remaining portion of biosample  12  may be removed after immobilization, such as by washing.  
         [0031]     As will be appreciated, the permanent electric dipole moment possessed by the ferroelectric material of transducer  14  may be reoriented by the application of an electric field. The effect of this field on transducer  14 , in turn affects the current/voltage across transducer  14 .  
         [0032]     A response signal, in this example case the voltage (V S ) across electrodes  20  and  22 , is detected using the signal detector, in this case voltmeter  26 . The response signal is indicative of the effect of the electric field in biosample  12  on transducer  14 . This voltage is compared with a reference voltage V R , which is the response voltage that would have been detected if biosample  12  were replaced with a reference sample while other conditions were substantially the same. The reference sample can be a blank sample or a sample of the sample type as biosample  12 . A blank sample is one that does not contain any target analytes. It may be advantageous if the blank sample is not electrically charged and has no or little electric polarization in an electric field. The reference sample can be a buffer solution such as de-ionized water. The reference voltage (V R ) can be measured simultaneously or sequentially with the sample voltage (V S ), using the same biosensor or separate biosensors. The reference voltage V R  may also be obtained from a previously conducted measurement, or from a database or a standard reference.  
         [0033]     As can be appreciated, it is possible to determine the concentration of the target analyte in biosample  12  if the analyte is of a known type. The concentration can be indicated by the difference between the sample response signal and the reference signal, which will be referred to herein as a signal shift, such as a voltage shift ΔV=V S −V R , when the current (I) is maintained constant. Similarly, when the voltage is the same for both biosample  12  and the reference sample, a current shift (ΔI) may result and the response signal can be the current and it is possible to detect analytes by establishing a constant voltage and detecting the current shifts.  
         [0034]     The signal shift may also be used to indicate the presence of different types of analytes as they may produce very different signal shifts.  
         [0035]      FIG. 4  shows an example line graph of voltage/current signal shift versus concentration of analyte within biosample  12 . Thus, reference voltages for a range of concentrations of the same type of analyte as the target analyte in biosample  12  can be obtained and tabulated or plotted, and the concentration in biosample  12  can be determined by matching measured V S  to table or plot. It is closest to the concentration corresponding to the V R  that matches V S  most closely in some situations, it may be necessary to measure a number of V S  at different conditions (e.g. different current levels) to determine the type of biosample  12 .  
         [0036]     It should be noted that other factors, such as temperature and the amount of biosample  12  or the analytes immobilized on sensor  10 , may also affect the signal shift. Thus, these conditions may need to be taken into account when comparing sample signal shifts.  
         [0037]     As should now be understood, while it may be possible to directly observe signal shifts between a biological sample and a reference sample without using a ferroelectric transducer, the presence of a ferroelectric transducer can enhance the signal shifts or make them easier to detect. Without being limited to a particular theory, one possible explanation for the enhancement is that a ferroelectric transducer can have a high dielectric constant and thus a high electric potential difference can be induced across the transducer when it is placed adjacent an electrically polarized sample. The polarized sample creates an external field in transducer  14  which polarizes transducer  14 . When biosample  12  contains analytes that are electrically polarized or charged under a potential bias, biosample  12  becomes electrically polarized. Usually, the higher the concentration of the analyte, the higher the polarization. Thus, the resulting signal shift can be more pronounced when a ferroelectric transducer and biosample  12  are placed adjacent to each other as compared to using no transducer or a non-ferroelectric transducer.  
         [0038]     To further illustrate, example relationships between voltage shifts and sample concentrations are shown in FIGS.  5  to  10 . Reference signals were obtained with a buffer solution containing de-ionized water. As can be seen in each figure, the voltage shifts are linearly dependent on the logarithm values of the concentrations of the analytes.  
         [0039]      FIGS. 5 and 6  show the results obtained with a Bovine Serum Albumin (BSA) solution as the biosample and a BST film as the transducer. For each measurement, 10 μl of BSA solution was dropped onto the BST film. A direct voltage was applied to top and bottom electrodes and was increased from 1 to 10 V with incremental of 20 mV. The voltage shifts were measured for different concentrations of BSA at a leakage current of 6 μA in  FIG. 5  and 0.4 μA in  FIG. 6 , respectively. The BSA concentrations were 1, 10, 20 or 30 mg/ml respectively for the data points shown in  FIG. 5  and from 1/512 to ½ mg/ml for  FIG. 6 .  
         [0040]      FIG. 7  shows the results obtained with immobilized BSA samples. The transducer used included a BST thin film. To immobilize the BSA sample, a thin layer of Au (about 200 nm thick) was coated on top of the BST thin film using an evaporation-beam method. The Au surface was cleaned by sonication for one hour in ethanol solution. 50 mg ProLinker™ B was dissolved in 60 ml of chloroform (CHCI 3 ) with a final concentration of 1 mM. The Au surface was immersed in the ProLinker™ B—CHCI 3  solution for one hour. As a result, a monolayer (SAM) of ProLinker™ B was formed on the Au surface through a self-assembling process. The final surface was rinsed with CHCI 3 , acetone, de-ionized water, ethanol, and then dried in a pure N 2  stream. The final surface was then immersed in a phosphate buffered saline (PBS) solution for one hour at room temperature, which had BSA concentrations of 10, 20 and 30 mg/mL respectively. Again, a direct voltage was applied to the top and bottom electrodes when about 10 μl di-ionized water was dropped onto the immobilized BSA surface. The leakage current was 6 μA.  
         [0041]      FIG. 8  shows the results for anti-BSA samples which were bound to immobilized BSA as described above where the concentration of the BSA was 10 mg/ml. For each measurement, 10 μl anti-BSA was dropped onto the immobilized BSA and was allowed to stay overnight. The sample surface was rinsed with di-ionized water and then dried by N 2 . A direct voltage was applied to the electrodes and the leakage current was 6 μA. The anti-BSA concentrations were 1/64, 1/16 and ¼ mg/ml respectively. The sensitivity for detection of anti-BSA concentration can be improved, for example, by choosing an optimized immobilized BSA concentration or a larger leakage current.  
         [0042]      FIG. 9  shows the results obtained with BSA samples being immobilized by covalent bonding with dodecyl phosphate (DDPO 4 ). In this case, the BSA was immobilized directly on a BST film. The BST surface was cleaned using O 2  plasma for three minutes and immersed in DDPO 4  solution for 48 hours. The surface was rinsed with di-ionized water and dried with pure N 2 . The BST surface was then immersed in a PBS solution containing BSA for one hour at room temperature, wherein for different measurements the solution had different BSA concentrations at 10, 20 and 30 mg/ml respectively. 10 μl di-ionized water was dropped onto the immobilized BSA surface and a direct voltage was applied. The leakage current was 30 μA. Compared with the results shown in  FIG. 8 , the detection sensitivity in this case has been improved, perhaps due to the larger leakage current.  
         [0043]      FIG. 10  shows the results obtained from anti-BSA samples which were bound to BSA immobilized on the BST film as described above. The leakage current was 6 μA. The selected concentration of immobilized BSA was 20 mg/ml. The anti-BSA concentrations were 1/64,¼, and 1 mg/ml.  
         [0044]     As now can be appreciated, biosensor  10  can be used to determine the types and concentrations of biological analytes in samples and can have some advantages over conventional biosensors. For example, it can have a simple structure, can be inexpensive, and can have high sensitivity and fast response time. Since transducer  14  can be formed using known techniques on a silicon wafer, biosensor  10  can be produced using currently available semiconductor techniques, which are mature and suitable for mass-production.  
         [0045]     Although only exemplary embodiments of this invention have been described above, those skilled in the art will readily appreciate that many modifications are possible therein without materially departing from the novel teachings and advantages of this invention. The invention, rather, is intended to encompass all such modification within its scope, as defined by the claims.