Abstract:
A bioelectronic microchip formed on a substrate ( 16 ) includes a plurality of field effect transistors ( 10 ), each including first ( 12 ) and second ( 14 ) electrodes on the substrate; and a channel ( 18 ) extending between the first and second electrodes. An organic semiconducting material fills the channel ( 18 ); and a dielectric layer ( 20 ) formed atop the first and second electrodes and the channel. An electrolyte ( 22 ) to hold a probe molecule may be formed on the dielectric. A third electrode ( 24 ) in proximity with the first and second electrodes and isolated therefrom contacts the dielectric. Capture of target molecules may be detected at each transistor through changes in source to drain characteristics. The method provides high density and low cost sensors, particularly in diagonistic and drug discovery applications.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application claims benefits from U.S. Provisional Patent Application No. 60/558,117 filed Apr. 1, 2004, the contents of which are hereby incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION  
       [0002]     The present invention relates generally to addressable biochips, and more particularly to chips incorporating field effect transistors and methods of use thereof. Embodiments of the invention can be used in assays to detect molecular interactions such as nucleic acid hybridization, protein binding or other chemical/electrochemical reactions.  
       BACKGROUND OF THE INVENTION  
       [0003]     Chemically modified field effect transistor (CHEMFET)-based sensor technologies have been studied as they have potential as microsensors for food, biomedical and environmental analytics. CHEMFETs have advantages over conventional ion-selective electrode-based sensors in terms of small dimensions, low-output impedance, fast response, mass-fabrication ability and great potential for integration into smart sensor arrays for detecting multi-analytes. However, present devices are mainly based on inorganic metal-oxide-semiconductor field effect transistors (MOSFETs). A limiting factor of such MOSFET devices is the relatively high manufacturing costs. This is particularly true for clinical applications which have strict safety requirements and where the use of disposable microsensors is highly desirable.  
         [0004]     Biochips, including DNA and protein array-based devices, have become an important tool in the life sciences, biomedical applications and drug discovery, due to the many benefits of miniaturization, integration and high-throughput mode of operation. Existing biochip technology is based however on fluorescent-based glass or silicon array fabrication and can be very expensive. For more than a decade now, organic field effect transistors (OFETs) based on conjugated polymers, oligomers, or other organic molecules have been envisioned as a viable alternative to more traditional, mainstream thin-film transistors (TFTS) based on inorganic materials.  
         [0005]     A major obstacle in the development of an addressable electronic array biochip is the occurrence of ionic shortage of different sensing arrayed electrodes because of the electrolytes in the sample. US 2002009064A1 discloses a high density addressable array biochip for electronic detection, which uses microchannels to separate different column arrays to eliminate the ionic shortage problem of addressed arrayed electrodes. The microfluidic method used however results in a much higher chip production cost and it is also very difficult to fabricate nano-array chips which require the nanoscale channels for eliminating the ionic shortage.  
         [0006]     There thus remains a need to develop more efficient devices and methods to fabricate electrical or electrochemical array chips. Particularly, there remains a need in the art to develop low cost column-and-row addressable biochip arrays for the electrical or electrochemical detection of molecular interactions that can be easily and cost-effectively fabricated and that reduces the cost of performing various analyses, while increasing the effectiveness and utility thereof. More particularly, there remains a need in the art to develop electronic addressable biochips.  
       SUMMARY OF THE INVENTION  
       [0007]     In accordance with an aspect of the present invention, a bioelectronic microchip includes: a substrate; a plurality of field effect transistors, each including: a first and second electrodes on the substrate; and a channel extending between the first and second electrodes; a semiconducting material filling the channel; a dielectric layer formed atop the first and second electrodes and the channel; a third electrode in proximity with the first and second electrodes and isolated therefrom.  
         [0008]     A solid or gel polymer electrolyte is coated atop of the dielectric layer. A potential applied to the third electrode exerts an electric field modulating the semiconducting channel of an associated transistor between the pair of electrodes through the solid/polymer electrolyte.  
         [0009]     In an embodiment, probe molecules of a certain type may be immobilized in the solid electrolyte on top of the dielectric layer. As a result, the modulation depth of the channel will depend on the electrical properties of the probe molecules. The probe molecules may be immobilized through entrapment or covalent binding thus allowing sensing target molecules through known chemical affinity or reactions.  
         [0010]     In an alternative embodiment, probe molecules of a certain type may be directly immobilized on the dielectric coating for sensing target molecules through chemical affinity or reactions. Again, a modulation depth of the channel depends on electrical properties of probe/target molecule combination.  
         [0011]     In an alternative embodiment, small covalent binding linker molecules can be grafted into dielectric layer by mixing the liker material and the dielectric material before forming the dielectric layer. Probe molecules of a certain type may be immobilized on the dielectric coating by covalent binding through the embedded linkers. Again, a modulation depth of the channel depends on electrical properties of probe/target molecule combination.  
         [0012]     The first and second electrodes act as drain/source electrodes, and the third electrode acts as a reference electrode (akin to the gate electrodes in a MOSFET).  
         [0013]     In accordance with another aspect of the present invention, the bioelectronic microchip may be all-printed and/or coated from solutions or/and inks to form an all-printed organic chemical field effect transistor with extremely low manufacturing and material costs.  
         [0014]     The plurality of field effect transistors may also be formed in a two dimensional array on the substrate. All first electrodes in a row are electronically interconnected (and may, for example be addressed as x 1 , x 2 ). All second electrodes in a column are electronically interconnected (and may for example be addressed as y 1 , y 2 , . . . ). All third electrodes may be electronically interconnected together as common line.  
         [0015]     The multiple transistors are thus x-y addressable. In an embodiment, a x-y addressable biochip forms to significantly reduce the multiplexing I/O lines in a high density array biochips.  
         [0016]     In accordance with another aspect of the present invention, an addressable bioarray, comprises a plurality of organic field effect transistors formed in a substrate, each of the field effect transistors including a source, and a drain; a channel formed therebetween and dielectric layer on the top of them. An electrolyte is formed atop the dielectric layer, to receive an analyte. A third electrode extends into the electrolyte. The presence of an analyte changes the channel characteristics under modulation of the third electrode, and may be detected through the soure/drain electrodes. Preferably, areas of the biochip other than those covered by the electrolyte are coated with a hydrophobic material to eliminate ionic shortage. The different columns/rows of electrodes are thus isolated by hydrophobic substrate, allowing the sample solution after the probes incubation with the target molecules to be blown out or drained out, for the subsequent electronic detection at different detection array spots.  
         [0017]     In accordance with another aspect of the present invention, there is provided a method of forming a biochip, by forming a plurality of transistors in a substrate.  
         [0018]     Each transistor may be formed by printing, coating, vacum deposition or otherwise forming parallel source and drain regions on a substrate separated by a gap. The gap defines a channel.  
         [0019]     Each channel is filled with an organic semiconduction material. A dielectric layer is coated on the top of the source/drain electrodes and is coated a solid or gel polymeric electrolyte to receive an analyte. Electrodes are formed in the electrolyte regions. The transistors are arranged in an array. Regions of the biochip that are not covered with electrolyte may further be covered with a hydrophobic coating. The size of each transistor is preferably in the nano-meter order.  
         [0020]     Additional source/drain electrodes extend from the transistors. Source/drain electrodes of transistors in rows are interconnected. The electrodes connected to the electrolytes of transistors in a column are also interconnected. This allows for row/column addressing of the organic chem./bio transistors. The channel may be filled by inkjet printing or chemical vaporation deposition method.  
         [0021]     In an embodiment, the organic transistor may be completely formed on an insulating bulk substrate, which can, for example, be made from materials such as plastic, ceramic, glass, paper, rubber, fabric, printed circuit board, silicon or combinations thereof. Conveniently, multiple CHEMOFETS may be formed in an addressable array. The addressable array electrode material can be, for example, solid or porous gold, silver, platinum, copper, titanium, chromium, aluminum, metal oxide, metal carbide, carbon, graphite, fullerene, conductive plastic, conductive polymer, metal impregnated polymers, nanocarbon tube, organic or polymer conducting wires or combinations thereof. All components of the biochip can thus be printed, including the organic transistor composed parts (conductors, source, drain, organic semiconductor and insulation layer) and the sensing transducer parts (reference electrode, polymer gel electrolyte).  
         [0022]     Other aspects and features 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  
       [0023]     In the figures which illustrate by way of example only, embodiments of the present invention,  
         [0024]      FIG. 1  is a schematic diagram of a chemically modified organic field effect transistor;  
         [0025]      FIG. 2  is a schematic diagram of a biochip, exemplary of an embodiment of the present invention;  
         [0026]      FIG. 3  is a plan view of an organic field effect transistor of the biochip of  FIG. 2 ;  
         [0027]      FIG. 4  is a schematic sectional view of a chemically modified organic field effect transistor of  FIG. 3 , along lines IV-IV;  
         [0028]      FIG. 5  is schematic process diagram illustrating formation of an exemplary electrolyte layer for use in an organic field effect transistor of the biochip of  FIG. 2 ;  
         [0029]      FIGS. 6A and 6B  are I-V curves illustrating an example biochip in use. 
     
    
     DETAILED DESCRIPTION  
       [0030]     The invention provides a chemically modified organic field effect transistor (CHEMOFET) based and column-and-row addressable array biochip. Embodiments utilize all printed OFETS in both the sensing and detecting components, combining molecular electronics (building a detection circuit with OFET) and bioelectronics (building an electronic biochip with OFET) to allow the manufacture of a high density array.  
         [0031]     An example single OFET  10  that may be used as a sensor is illustrated in  FIG. 1 . As illustrated, source and drain electrodes  12 ,  14  are formed in a semiconducting substrate  16 . A channel  18  is formed between source and drain electrodes  12 ,  14 . A dielectric insulating layer  20  is formed atop channel  18 . Layer  20  is coated with an electrolyte  22 . A reference electrode  24  contacts electrolyte  22  as a substitute for a conventional gate electrode. Probe molecules (not shown) are immobilized in dielectric layer  20  or in electrolyte  22  on top of the dielectric layer  20 . Probe/target molecule interaction may change the electrical properties of the dielectric layer  20  thus allowing for the sensing of target molecules in samples. Different analytes or different concentrations of the same analyte in the electrolyte can modify dielectric layer  20  of OFET  10 , resulting in different changes of density of electrons in the inversion layer built by imposing voltage between the reference electrode and the source electrode. Typically, the changes very sensitively correspond to the concentration of the analyte.  
         [0032]      FIG. 2  schematically illustrates an exemplary source/reference electrode addressable OFET array biochip  100 . Chip  100  includes a plurality of OFETs  30 , similar to OFET  10  of  FIG. 1 , arranged in rows and columns on a substrate  102 . As will become apparent, OFETs  30  may be printed or vapour deposed on substrate  102 . For nanoscale printing, atomic force microscopy may be used as tool for the nanoprinting.  
         [0033]     Enlarged plan and cross-sectional schematic views of each individual OFETs  30  are illustrated in  FIGS. 3 and 4 . As illustrated, each OFET  30  includes source and drain electrodes  32  and  34  spaced from each other. An organic semi-conducting material is formed in the space between source and drain electrodes  32 ,  34  to form a conductive channel  38  therebetween. The organic semiconducting material may, for example, be selected from polythiophene, polyacetylene, phthalocyanine, poly(3-alkylthiophene), α,ω-hexathiophene, pentacene, α,ω-dihexylhexathiophene, poly(thienylene vinylene), C60, bis(dithienothiophene), α,ω-dihexylquaterthiophene, dihexylanthradithiophene, fluorinated NTCDI, α,ω-dihexylquinquethiophene, or their combinations. A dielectric insulator  40  at least partially covers channel  38 . An electrolyte layer  42  is formed atop insulator  40 . As will be appreciated the depth of channel  38  may be controlled by the voltage applied to electrolyte  42 .  
         [0034]     A reference electrode  36  is formed next to, and in proximity with source and drain electrodes  32 ,  34 , and is covered with electrolyte  42 .  
         [0035]     Reference electrodes  36  of all OFETs  30  within a column are interconnected. A single reference electrode thus serves all source and drain electrodes  32 ,  34  of OFETs  30  in the same column. Similarly, all source electrodes  32  within a row are interconnected to each other, and all drain electrodes  34  within a column are interconnected.  
         [0036]     Electrolyte  42  may be solid or gel, and may for example be polyacrylamide. Electrolyte  42  acts as a receptacle for analyte solutions. Electrolyte  42  is pattern-coated or deposited in such a way that the electrolyte  42  of each OFETs  30  does not overlap with the electrolyte of adjacent OFETs  30 . This is best illustrated in  FIG. 2 .  
         [0037]     The remaining surface of biochip  100  is coated with a hydrophobic layer (not shown). The hydrophobic layer is much thinner than electrolyte  42 , and therefore allows only the electrolyte  42  or dielectric layer  40  to be exposed.  
         [0038]     In this embodiment, OFETs  30  are printed onto the insulating bulk substrate, which can, for example, be made from materials such as plastic, ceramic, glass, rubber, fabric, printed circuit board, silicon or combinations thereof. The electrode material can be, for example, solid or porous gold, silver, platinum, copper, titanium, chromium, aluminum, metal oxide, metal carbide, carbon, graphite, fullerene, conductive plastic, conductive polymer, metal impregnated polymers or combinations thereof. As will become apparent, all components of biochip  100  can be printed, including the components of OFETs  30  (conductors, source, drain, organic semiconductor and insulation layer) and reference electrode, and polymer gel electrolyte.  
         [0039]     As schematically illustrated in  FIG. 5 , probe molecules that bind to specific target molecules, may be embedded in, or attached to, electrolyte  42 . Electrolyte  42  is thus used as both an ionic conductor and sensing transducer, to interact with analyte applied to the surface of biochip  100 . Electrolyte  42  can be used as the matrix for universal bioconjugation to immobilize probe molecules. In one embodiment a certain percentage of streptavidin molecules may be mixed into a polymer gel forming electrolyte  42 , which for example can be polyacrylamide gel that is porous, followed by UV crosslinking, which makes the gel insoluble and which can also function as an electrolyte.  
         [0040]     As another example, biotinylated protein or DNA probes can be directly attached to the gel electrolyte  42  by biotin-streptavidin conjugation, as shown in  FIG. 5 . Only the section of the gel on the insulation layer  40  that contains streptavidin, which can be made by separately printing the gel electrolyte to overlap both the insulation layer and the reference electrode, is shown.  
         [0041]     Alternatively, cross-linked polyacrylamide can be directly immobilized with probes by reacting the amino groups of lysine with NHS esters, providing a biocompatible aqueous environment for protein interactions through gel aldehyde groups and amino groups of the probe proteins.  
         [0042]     Biochip  100  can thus be used to detect molecular interactions such as nucleic acid hybridization, protein binding or other chemical/electrochemical reactions.  
         [0043]     In a further embodiment, the probe molecules may be directly immobilized on dielectric insulation layer  40  before electrolyte coating. The immobilization may be conducted by adding bioconjugation linker molecules into dielectric materials. In this case, layer  42  coating is thinner and more porous for target molecule access.  
         [0044]     Of course, each OFET  30  on a biochip  100  may include its own (and thus possibly different) pre-selected probe molecule.  
         [0045]     In operation, target molecules in sample analyte (typically in a solution) that may interact with the probe molecules are placed on the electrolyte  42  at individual OFETs  30 . Analyte, if present, may be immobilized on electrolyte  42  by complementary probe molecules. The sample solution may be drained to facilitate electronic detection of the analyte at the different detection array spots. As only the insulation layer  40 , reference electrode  36  and the coated solid or gel electrolyte  42  is exposed to the sample, and all other components and surfaces (such as source/drain electrodes and conductors) are insulated with the hydrophobic coating, ionic shortage is prevented, making detection very simple. Conveniently, leakage current is reduced by the coating hydrophobic polymer layer. This, in turn, enhances modulation of current through channel  38  in the presence of analyte. Additionally, a hydrophobic surfactant may be used to reduce moisture adsorption. The hydrophobic layer covering all exposed current connectors or part of electrode surface also prevents possible gas during OFET measurement. The organic semiconductor and insulation material should also be chemically compatible to reduce interfacial resistance.  
         [0046]     As will be appreciated, the presence of trapped analyte at an OFETs  30  will affect the conductivity of a channel  38  beneath insulation layer  40  and electrolyte  42  of that OFET  30 . This change in conductivity may be detected by detecting changes source to drain conductivity for applied voltage. That is, source and drain voltages may be individually applied to each source/drain pair and resulting current flow may be measured. This can be conducted by simply multiplexing switches (not shown) to different source/reference electrode addresses, such as S 1 R 1 , S 2 R 2  etc for detection at different array spots (where S 1 , S 2 , . . . represent source electrode addresses and R 1 ,R 2 , . . . represent the reference electrode addresses as shown in  FIG. 2 ). Use of addressable array chips significantly reduces the number of I/O lines for much simpler multiplexing, thus further reducing the manufacturing cost of the detection system.  
         [0047]     Advantageously, embodiments of the present invention utilise an addressable platform. As well, embodiments of the present invention do not require the fabrication of microchannels to prevent ionic shortage as this is prevented by the hydrophobic coating.  
         [0048]     Conveniently, biochip  100  can be manufactured at a low cost. All-printed OFETs may be formed using a conventional bottom-up fabrication process with inexpensive materials. Initially, source drain and reference electrodes  32 ,  34  and  36  may be printed or vapour deposed, or laser cutting in metal coated isolated substrate. Organic semiconductor material may be printed or vapour deposed between source and drain electrodes. Thereafter, insulating regions  40 , electrolyte  42  and hydrophobic layer may be formed on the printed electrodes and gates. Exemplary embodiments may be formed as micro- and nano-scale biochips, as the organic inks can be tailored for nano-printing with imprinting, AFM assist and/or self-assembly process. Each OFET  30  may thus be formed, for example, with dimensions less than 200 μm×200 μm and typically between 10 nm×10 nm to 200 μm×200 μm. This allows miniaturisation to make portable electronic detection devices. The nano-scale OFETs can thus be used to build array sensors or wireless networking bio/chemical sensors.  
         [0049]     Embodiments of the present invention further provide a biochip with a universal bioconjugation method for the immobilization of multi-biomolecular probes, which has so far not been used for field effect transistors or OFETs.  
         [0050]     As illustrated, exemplary embodiments provide an electronic detection method for biochips that is based on the modulation of the field effect transistor, which is a label-less method. The label-less method is important in protein array chips where the proteins are very difficult to label, for example low molecular weight protein molecules without an additional epitope for biomolecular labelling.  
         [0051]     Electrical or electrochemical detection of molecular interactions, as used in biochip  100  also require less expensive detection systems and less instrumentation than detection techniques relying on radioactive or fluorescent labels that require photonic sensors.  
         [0052]     Optionally biochip  100  may be plasma treated to reduce leakage current at OFETs  30 . For example a biochip  100  once formed may be subjected to plasma surface treatment as follows: 
        1. The surface of the substrate may be cleaned, using for example, flowing nitrogen gas.     2. The substrate may be dried (e.g. in a vacuum at 120 C for 1 hour).     3. Plasma Enhanced Chemical Vapour Deposition (PECVD) may be used to treat the biochip (e.g. under Ar 150 W for 1 min in a PECVD system).     4. The biochip may be plasma treated—by, for example, applying NH 3  plasma at 150 W for 10 mins by placing the biochip in a NH 3  purged plasma chamber.     5. Cast the P3HT-Toluene solution onto the substrate to cover the gap between source and drain.     6. Then placed the biochip with P3H-T in an oven to dry and anneal the biochip at 70 C for 1 hr.        
 
         [0059]     I-V curves at individual OFETs were measured with and without plasma treatment, as shown in  FIGS. 6A and 6B . Before treatment with plasma, the organic transistor had poorer modulation shown in  FIG. 6A . After plasma-treatment, the transistor performance was improved. Measurement also demonstrated the leakage current of OFETs was much reduced in comparison to the biochip without plasma treatment. The Plasma treatment could remove some conductive impurity on the surface components of the biochip, this could give better modulation and reduce the leakage current.  
         [0060]     As should now be appreciated, biochip  100  has various applications, for example in clinical diagnostics, drug discovery, food safety, and environmental protection.  
         [0061]     Of course, the above described embodiments are intended to be illustrative only and in no way limiting. The described embodiments of carrying out the invention are susceptible to many modifications of form, arrangement of parts, details and order of operation. The invention, rather, is intended to encompass all such modification within its scope, as defined by the claims.