Patent Abstract:
A biochip for detecting or sequencing biomolecules and a method of making the same. The biochip comprises a base member; a dielectric layer being deposited on the base member and having at least two rows of discrete recesses being formed thereon; and two or more electrodes being sandwiched between the base member and the dielectric layer and running under respective row of discrete recesses, the two or more electrodes are separated from each other along length by a portion of the dielectric layer; wherein the dielectric layer defines a continuous operation surface above the electrodes and on which the discrete recesses are deposited for detecting or sequencing of biomolecules, when an electric field is applied through the electrodes, a field gradient is created to draw biomolecule towards a preferred part of the operation surface.

Full Description:
BACKGROUND OF THE INVENTION 
       [0001]    The present invention relates to a biochip for the determination of the concentration of one or more types of specific biomolecules in a sample and its fabrication, for example particularly, but not exclusively, a biochip and its fabrication. 
         [0002]    Nowadays, fast and high throughput biomolecular based detection devices tend to be miniaturized and high-level of integration. A general term for such devices is called “biochip”. A biochip composes of a substrate for identifying the target from the sample solution being dispensed onto the substrate. Various sensing parts or the biomolecular reactants are facilitated on the substrate depending on specific applications. To enhance the in situ biochemical reaction efficiency, a close distance and locally high concentration of the reactant are both necessary. Therefore, efforts have been made to actively transport and replenish the target molecules, because these movements will increase opportunities for the target entities to meet/react with their counterparts, i.e. probe entities/biomolecules, which are normally immobilized on the substrate. Electrokinetics (EK) technologies have been developed and widely used as a mechanical-part-free, flexible and highly programmable tool for fluid and microparticle manipulation, especially in Lab-On-A-Chip (LOAC) platforms. 
         [0003]    Currently, there are some limitations in EK manipulation in LOAC devices. Under DC electric field regime, electrophoresis (EP) technology has been widely applied, such as the well-known gel electrophoresis. In general, DC electric field is only effective for either positively or negatively charged entities at a time. For biomolecules having complex conformations and charge conditions, it is difficult to manipulate EP transportation with good consistency and efficiency. Besides, even the highly corrosion-resistant electrode, like Platinum (Pt), will degrade with the operation time, which would also affect the accuracy and efficiency of such devices. When using AC electric field, dielectrophoresis (DEP) have become an outstanding technology in manipulating any charged and neutral micro-particles. The major drawback of DEP is the short effective range, while other AC electrohydrodynamic (EHD) effects have been proven capable of driving fluids in longer range. However, the fluid flow is not efficient for confining and concentrating small molecules like nucleic acids. 
         [0004]    Therefore, we seek to provide a comprehensive and robust solution to increase the efficiency of biochemical reaction through the conceptual design of device structures and fabrication method for combinational EK manipulations. 
       SUMMARY OF THE INVENTION 
       [0005]    According to the invention, there is provided a biochip for detecting or sequencing biomolecules comprising:
       a base member;   a dielectric layer being deposited on the base member and having at least two rows of discrete recesses being formed thereon; and   two or more electrodes being sandwiched between the base member and the dielectric layer and running under respective row of discrete recesses, the two or more electrodes are separated from each other along length by a portion of the dielectric layer;   wherein the dielectric layer defines a continuous operation surface above the electrodes and on which the discrete recesses are formed for detecting or sequencing of biomolecules, when an electric field is applied through the electrodes, a field gradient is created to draw biomolecules towards a preferred part of the operation surface.       
 
         [0010]    Preferably, the recesses comprise one or more well and furrow formed between two rows of wells. 
         [0011]    More preferably the furrow separates the two or more electrodes. 
         [0012]    Advantageously, each electrode extends across the base member and is common to the wells in a same row. 
         [0013]    More advantageously, the electrode comprises an elongate stripe of electric conducting material. 
         [0014]    Preferably, the electrode is deposited underneath the dielectric layer. 
         [0015]    Preferably, the electrode has a predetermined width of about 10 nm to 1 mm. 
         [0016]    More preferably, the electrode has a predetermined thickness of about 1 nm to 1 mm. 
         [0017]    Yet more preferably, the dielectric layer has a thickness of about 1 nm to 1 mm. 
         [0018]    It is preferable that each well has a predetermined width of about 10 nm to 1 mm and has to be smaller than the predetermined width of the electrode as described above. 
         [0019]    It is advantageous that each well has a predetermined depth of about 1 nm to 1 mm and has to be smaller than the thickness of the dielectric layer as described above. 
         [0020]    Preferably the furrow has a predetermined width larger than twice the thickness of the dielectric layer as described above and smaller or equal to the width of the electrode as described above. 
         [0021]    Preferably, the dielectric layer comprises a dielectric material selected from a group consisting of silicon oxide, silicon nitride, aluminum oxide and titanium oxide. 
         [0022]    More preferably, the base member comprises a layer of thermal oxide deposited on a layer of silicon substrate. 
         [0023]    Yet more preferably, one or more probe entities are attached to the operation surface in the recesses for catering the use of a low frequency electric field to create a field gradient that draws target entities towards the recess. 
         [0024]    It is preferable that one or more probe entities are attached to the operation surface and outside of the recesses for catering the use of a high frequency electric field to create a field gradient that forces target entities towards operation surface outside the recess. 
         [0025]    In another aspect of the invention there is provided a method of fabricating the biochip as described above comprising the steps of:
       a) providing a base member;   b) photomasking an upper surface on the base member;   c) etching two or more recesses on the upper surface;   d) depositing a dielectric material on an upper layer over the two or more recesses;   wherein the two or more recesses define a contour on the upper surface, the dielectric material adopts the contour and hardens to form a contoured operation surface.       
 
         [0031]    Preferably, the base member comprises a metal layer deposited on a thermal oxide layer, the metal layer defines the upper surface on which a first round of the steps b) and c) are conducted to form the two or more recesses. 
         [0032]    More preferably, a second round of steps b) and c) are conducted on the upper surface and between the two recesses to create a further recess therebetween which defines the upper layer on which step d) is performed. 
         [0033]    Yet more preferably, the step d) is conducted after the first round of steps b) and c) to form a further upper surface, thereafter a second round of steps b) and c) are conducted on the further upper surface to further define the contour and form the upper layer. 
         [0034]    Advantageously, the second round of steps b) and c) create one or more further recesses between the two or more recesses formed by the first round of steps b) and c). 
         [0035]    More advantageously, the base member includes at least a thermal oxide layer which defines the upper surface and a first round of steps b) and c) are conducted on the upper surface to define the contour. 
         [0036]    It is advantageous that the method further comprising the step of depositing a layer of metal onto the upper surface to form a further upper surface which adopts the contour. 
         [0037]    Preferably, wherein a second round of steps b) and c) is conducted on the further upper surface to create one or more further recesses between the two recesses to further define the contour and form the upper layer. 
         [0038]    More preferably, a step d) is conducted on the upper layer such that the dielectric material adopts the further defined contour and hardens to form a contoured operation surface. 
         [0039]    In a further aspect of the invention there is provided a method of fabricating the biochip comprising the steps of:
       a) providing a base member which includes a metal layer deposited on a thermal oxide layer;   b) photomasking an upper surface on the metal layer;   c) etching two or more recesses on the upper surface;   d) depositing a dielectric material on the upper surface forming an upper layer over the two or more recesses;   e) photomasking the upper layer;   f) etching a recess on the upper layer;   g) depositing a dielectric material on the upper layer over the recesses;   wherein the recesses define a contour on the upper surface and the upper layer, the dielectric material adopts the contour and hardens to form a contoured operation surface.       
 
         [0048]    In an even further aspect of the invention there is provided a method of fabricating the biochip comprising the steps of:
       a) providing a base member which includes a metal layer deposited on a thermal oxide layer, the metal layer defines the upper surface on which a first round of steps b) and c) are conducted to form the two or more recesses;   b) photomasking an upper surface on the base member;   c) etching two or more recesses on the upper surface;   d) conducting a second round of steps b) and c) on the upper surface and between the two recesses to create a further recess therebetween which defines an upper layer;   e) depositing a dielectric material on an upper layer over the recesses;   wherein the recesses define a contour on the upper layer, the dielectric material adopts the contour and hardens to form a contoured operation surface.       
 
         [0055]    In another aspect of the invention there is provided a method of fabricating the biochip comprising the steps of:
       a) providing a base member which includes at least a thermal oxide layer which defines the upper surface and a first round of steps b) and c) are conducted on the upper surface;   b) photomasking an upper surface on the base member;   c) etching a recess on the upper surface;   d) depositing a layer of metal onto the upper surface to form a further upper surface which adopts the contour of the upper surface;   e) a second round of steps b) and c) is conducted on the further upper surface to create further recess on either side of the first formed recess to further define the contour and form the upper layer   d) depositing a dielectric material on an upper layer over the recesses;   wherein the dielectric material adopts the contour on the upper layer and hardens to form a contoured operation surface.       
 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0063]    The invention will now be more particularly described, by way of example only, with reference to the accompanying drawings, in which: 
           [0064]      FIG. 1A  is a perspective view of the biochip in accordance with the invention; 
           [0065]      FIG. 1B  is a schematic cross-sectional representation of the biochip in  FIG. 1A ; 
           [0066]      FIG. 2  is a schematic side view of part of the biochip in  FIG. 1A  showing a recess with probe entities attached thereto on an operation surface and an electrode; 
           [0067]      FIG. 3  is a schematic representation of a part of the biochip in  FIG. 1A  with indications showing a long range fluid transport and a short range fluid transport or localized regulation; 
           [0068]      FIGS. 4A to 4D  are schematic representations of a EK operation of a circular recess in the biochip of  FIG. 1A . Specifically,  FIG. 4A  shows the circular recess without EK operation,  FIG. 4B  shows the EK operation of the circular recess under a low frequency AC electric field which is applied across electrodes of the biochip, indications are provided to show direction of the long range fluid transport and the short range fluid transport or localized regulation,  FIG. 4C  shows the EK operation under a medium frequency of AC electric field applied across the electrodes of the biochip, indications are provided showing the direction of the long range fluid transport and the short range fluid transport or localized regulation,  FIG. 4D  shows the EK operation under a high frequency of AC electric field applied across the electrodes of the biochip, indications are provided to show the direction of the long range fluid transport and the short range fluid transport or localized regulation; 
           [0069]      FIG. 5  is a schematic illustrative comparison between binding of target and probe entities via passive diffusion and that via electrokinetically enhanced transportation; 
           [0070]      FIG. 6  is a schematic illustration of a first embodiment of a fabrication process of the biochip in  FIG. 1A ; 
           [0071]      FIG. 7  is a schematic illustration of a second embodiment of the fabrication process of the biochip in  FIG. 1A ; 
           [0072]      FIG. 8  is a schematic illustration of a third embodiment of the fabrication process of the biochip in  FIG. 1A ; 
           [0073]      FIG. 9A  is a schematic illustration of the electric connection of the biochip in  FIG. 1A ; 
           [0074]      FIG. 9B  is a photographical illustration of the biochip in  FIG. 9A  showing distribution of suspended target entities on the biochip at different frequencies of electric field applied across the electrodes; 
           [0075]      FIG. 10  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, in a medium on the biochip under an AC electric field at a frequency of 1 kHz; 
           [0076]      FIG. 11A  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around the recess in the biochip in a low conductivity medium under an AC electric field of low frequency at 1 kHz; 
           [0077]      FIG. 11B  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around a gap region between two recesses in the biochip of  FIG. 11A  in a low conductivity medium under an AC electric field of low frequency at 1 kHz; 
           [0078]      FIG. 12A  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around the recess in the biochip in a low conductivity medium under an AC electric field of medium frequency at 10 kHz; 
           [0079]      FIG. 12B  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around a gap region between two recesses in the biochip of  FIG. 12A  in a low conductivity medium under an AC electric field of medium frequency at 10 kHz; 
           [0080]      FIG. 13A  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around the recess in the biochip in a low conductivity medium under an AC electric field of high frequency of 1 MHz; 
           [0081]      FIG. 13B  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around a gap region between two recesses in the biochip of  FIG. 13A  in a low conductivity medium under an AC electric field of high frequency of 1 MHz; 
           [0082]      FIG. 14A  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around the recess in the biochip in a high conductivity medium under an AC electric field of low frequency of 1 kHz; 
           [0083]      FIG. 14B  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around a gap region between two recesses in the biochip of  FIG. 14A  in a high conductivity medium under an AC electric field of low frequency of 1 kHz; 
           [0084]      FIG. 15A  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, around the recess in the biochip in a high conductivity medium under an AC electric field of high frequency of 1 MHz; 
           [0085]      FIG. 15B  is a schematic illustration of the electric field distribution and the direction of DEP force, shown by arrows, at an area around a gap region between two recesses in the biochip of  FIG. 15A  in a high conductivity medium under an AC electric field of high frequency of 1 MHz; 
           [0086]      FIG. 16  is a photographical illustration of a part of the biochip in  FIG. 1  demonstrating a comparison between the concentration of bound target to probe biomolecules under passive diffusion and the concentration of bound target to probe biomolecules under electrokinetically enhanced transportation for a same period of time; 
           [0087]      FIG. 17  showing the pattern of immobilized probe biomolecules and the meaning of the probe biomolecules in the region of the experimented biochips shown in  FIG. 16 ; 
           [0088]      FIG. 18  is a bar chart comparing binding efficiencies represented in artificial unit intensity of electrokinetically enhanced transportation and binding efficiencies represented in artificial unit intensity of passive diffusion; and 
           [0089]      FIG. 19  is a schematic representation showing various dimension of the biochip. 
       
    
    
     DETAILED DESCRIPTION OF EMBODIMENTS 
       [0090]    Referring to  FIGS. 1A and 1B  of the drawings, there is shown an embodiment of a biochip  100  in accordance with the invention. Two or more discrete wells/recesses  101  are formed on an operation surface  108 A. These wells/recesses are aligned to form a plurality of rows of wells/recesses  103 . These rows  103  run parallel to one another and extend across the biochip  100 . Each row  103  is separated by a recess in the form an elongated furrow  104 . The shape of the wells/recesses  103  can be of any size and shape. 
         [0091]    The term recess may be used to describe any indentation including wells  103  and furrow  104 . 
         [0092]    As shown in  FIG. 1B , the biochip  100  is a lamination of or an array of layers of material. It has a base member which may include a layer of thermal oxide  105  deposited on a layer of silicon substrate  106 . Stripes of conducting material  107  is placed on the layer of thermal oxide  105  forming the electrodes  107  of the biochip  100  that runs underneath each row  103 . A layer of dielectric material  108  is deposited on the electrodes  107  to form an operation surface  108 A of the biochip. The electrodes  107  are separated from one another by the furrow  104 . The discrete wells/recesses  101  and the furrow  104  are formed on the operation surface  108 A in the form of a concave structure and a groove respectively. In other words, the operation surface is a contoured operation surface  108 A. Probe entities  109  are attached to the operation surface  108 A in conventional manner. 
         [0093]    The biochip  100  when in use is covered by an aqueous solution with target entities disbursed therein which forms the medium for EK operation. The electrode arrays  107  is embedded under insulating layers such as the layer of dielectric material  108 . This insulating layer prevents electrode from direct contact with the medium solution, leading electrochemical reaction, namely, electrolysis. By applying time varying electric field, the electric field is able to penetrate into the solution medium above the insulating surface. The existence of electric field strength in the medium can induces electrokinetic fluid flow and particle movement. On the surface of insulating layer  108 , wells/recesses structures are provided to specifically regulate EK effects and concentrate target entities to the reaction sites where the probe entities are immobilized. With the increased local concentration, the opportunity of collision between target entities with the corresponding probe entities is enhanced. This process is achieved by DEP forces and ACEO/ACET fluid flow on the chip surface, and is far more efficient than passive diffusion in conventional reaction devices. An example is shown in  FIGS. 16 and 18  and will be described later. 
         [0094]    The biochip  100  is capable of manipulating fluid flow and assists surface focusing efficiency. The electrokinetic forces acting on target entities, such as EP and DEP, strongly rely on the field strength and the gradient of field strength square, respectively. However, the electric field strength attenuates exponentially into the medium, which confines the effective range of direct EK force on particles within a very short distance above electrode  107  surface. In this case, electrode array  107  is in planar manner, which is designated to induce long range fluid flows by ACEO and/or ACET effects under AC electric field. The ACEK induced fluid flows are patterned in a circulating manner, which can continuously refresh target entities in surface fluid with those in the bulk fluid. 
         [0095]    Finally, by applying AC electric field to the biochip  100 , target entities suspended in an aqueous solution are driven to the reaction sites on the chip surface via induced fluid flows. The surface structures further regulate the flow and concentrate the target entities to designated reaction sites with the assistance of DEP forces. The fluid flow circulation, in the meantime, acts to replenish the target entities from the bulk solution. Thus, with the combination of EK effects, both long range and short range manipulation are achieved. The resulting reaction efficiency can be enhanced from diffusion-based mechanism. 
         [0096]    In more detail, as shown in  FIG. 19 , width (B) of the discrete well/recess  101  is about 10 nm to 1 mm and would be smaller than the width (F) of the electrode  107  which is about 10 nm to 1 mm. Depth (A) of the well/recess  101  is about 1 nm to 1 mm and is smaller than thickness (G) of the dielectric layer  108  which is about 1 nm to 1 mm. The width (D) of the furrow  104  is larger than twice the thickness (G) of the dielectric layer  108  and smaller or equal to the width (F) of the electrode  107 . The depth (C) of furrow  104  is equal to the thickness (E) of the electrode  107  which is about 1 nm to 1 mm. The discreet wells/recesses  101  have sharp upper rims with edge radius that is equal to the excess length of the thickness (G) of the dielectric layer  108  over the depth (A) of the well/recess  101 . The discreet furrows  104  also have sharp upper rims with an edge radius which is equal to the thickness (G) of the dielectric layer  108 . In summary, the smaller the edge radius, the greater an electric gradient is established in the medium adjacent the rim and edge when electric field is applied to the electrode. 
         [0097]    CMOS fabrication techniques and processes for wafer-level production are used to create the biochip  100 . There are four crucial layers. The biochip substrate is silicon wafer  106 . Thermal oxidation is performed to create a foundation layer, a thermal oxide layer  105  for metal deposition. Sputtering method is used to form a metal layer  107  and pattern it into arrays of microelectrodes by photolithography. Then a dielectric layer  108  is deposited on the metal layer  107  for protecting the electrodes  107  and insulation. The wells/recesses  101  and furrows  104  are formed by photomasking and etching by photolithography. The materials of the dielectric layer will be detailed below. 
         [0098]    The silicon substrate  106  is preferably a substrate of silicon based materials and solid polymers materials. The layer of dielectric material  108  is preferably an insulating layer made of silicon oxide, silicon nitride, titanium oxide or other dielectric materials. 
         [0099]    Now we introduce the mechanism and controlling conditions of EK manipulation on the biochip. 
         [0100]    The array of electrodes  107 , more preferably microelectrodes, are embedded in the silicon substrate  106  preferably a silicon chip to create non-uniform distribution of electric field in the solution medium. Multiple EK effects due to non-uniform electric field are responsible for concentrating target entities, inducing fluid flow/enabling circulation above the biochip  100 . Micro- or submicro-scaled target entities, such as biomolecules for example nucleic acid, suspended in the solution medium are being transported and circulated close to the operation surface  108 A from the solution medium due to the electric field generated from the array of electrodes  107 . Generally, the large-scale non-uniform electric field is created at the furrow  104  between each electrode which can generate long range EK fluid flow and transport target entities in the bulk to the region close to the surface of the chip. The contoured operation surface  108 A, particularly with the wells/recesses  101 , modifies local electric field distribution and enhance short region EK performance. 
         [0101]    The major effective EK activities include Dielectrophoresis (DEP), AC Electroosmosis (ACEO), and AC Electrothermosis (ACET). The overall phenomenon is always a combination of multiple effects. ACEO and ACET are categorized as electrohydrodynamic effects, which induces long range fluid flow in the solution medium. DEP is short-ranged motion on particles. As illustrated in  FIG. 4 , ACEO and ACET are responsible for generating fluid flow for long range transportation  110 , and DEP takes part in short range confinement/short range fluid transport/localized regulation  111 . With induced long range fluid flow  110 , particle-like entities including charged or uncharged particles, sized from micron to submicron can be transported effectively close to the operation surface  108 A. The wells/recesses  101  patterned on the operation surface  108 A further regulates the local electric field and thus the target particles close to the operation surface  108 A is concentrated by DEP forces inside the wells/recesses  101 . 
         [0102]    In more detail, ACEO arises due to the interaction between the electric double layer (EDL) formed at the interface between a solution and a charged solid surface like the operation surface  108 A and an electric field in the tangential direction (i.e. E t ) to drive the ions in the diffuse layer of the EDL. When a pair of planar electrodes is charged with opposite polarity, the electric field is stronger at the gap and weaker at the electrode centre, therefore the E t . The certain circumstances, there exist another electrohydrodynamic effect, known as ACET, which is due to the interaction between the electric field and the gradient of fluid properties. Since AC electric field can induce Joule heating in the solution medium and is more significant in the region of high field strength, the regional temperature change induces variation of fluid density, and thus conductivity and permittivity. For a planar electrode pair, ACET can form circulation from the electrode gap, and stir the bulk fluid in micro-scaled range. In summary, long range fluid flows can be generated using various classical electrode patterns, such as the parallel, castellated, quadrupole etc. 
         [0103]    For short range manipulation, DEP effects become more promising as the field strength as well as its gradient are high. The structure that can induce sharp field gradient is the edges of wells/recesses  101  being patterned on the operation surface  108 A. This localized force field enables designated driving patterns for short range collection  111  of the target entities. 
         [0104]    Furthermore, it is possible to control patterns of fluid flow and particle collection by changing the applied voltage, frequency and formation of the AC electric field. The mechanism can be used for enhancing biochemical reaction efficiency between target molecules suspended in the solution medium and counterpart molecules e.g. probe entities  109  immobilized on the operation surface  108 A. 
         [0105]    The contoured operation surface  108 A above the electrode array  107  generates gradients of electric field and alters DEP, ACEO and ACET at specific. Depending on the frequency of the electric field and the conductivity of the solution medium, direction of the long range transportation  110  as well as that of the short range confinement/short range fluid transport/localized regulation  111  can be manipulated. Voltage is more related to the overall strength of EK effects, i.e. DEP force is proportional to Δ|E 2 | in magnitude. 
         [0106]      FIG. 4A to 4D  is a schematic illustration of the direction of the long range EK flow  112  and the short range EK flow  113 , hence the particle concentration inside and outside the well/recess  101  when different voltage, frequency and formation of the AC electric field is applied to the electrode array  107 . The long and short range EK flow directly constitute the long range transportation  110  and short range confinement/short range fluid transport/localized regulation  111  respectively for transporting the target entities in the solution medium. In  FIG. 4A , show a top plan view of the well/recess  101  on the contoured operation surface  108 A.  FIG. 4B  shows the direction of the EK flows  112 / 113  when a low frequency electric field is applied. The main stream of EK flows  112 / 113  are pointing to the centre of the electrode, and the localized gradient at the edge of well/recess  101  directs particles towards the centre of the well/recess  101  and the electrode  107 .  FIG. 4C  shows the direction of the EK flows  112 / 113  when a medium frequency electric field is applied. The main stream of EK flow  112  are pointing to the centre of the electrode  107 , and the localized gradient  113  at the edge of well/recess  101  directs particles away from the centre of the well/recess  101  and the electrode  107 .  FIG. 4D  shows the direction of the EK flow  112 / 113  when a high frequency electric field is applied. The main stream of EK flow  112  and the localized gradient  113  at the edge of well/recess  101  directs particles away from the centre of the well/recess  101  and the electrode  107 . As adjacent wells/recesses  101  are separated by a furrow  104 , when the main stream EK flow  112  and the localized gradient  113  are directed away from the centre of the well/recess  101 , they are directed towards the furrow  104 . The situation in  FIG. 4B  is most optimal when the probe entities  109  are attached inside the well/recess  101 . The condition in  FIGS. 4C and 4D  is most optimal when the probe entities  109  are attached to the operation surface  108 A outside the well/recess  101  or in the furrow  104 . 
         [0107]    With the contoured operation surface  108 A, we are able to manipulate target entities collection pattern on the operation surface  108 A using different frequencies, voltages and formations of the AC electric field. We are able to pattern the operation surface  108 A in matrix or in any asymmetric arrangement on the electrodes  107 , depending on specific applications. 
         [0108]    As shown in  FIG. 9A  there is an example of an operation surface with an N by N well/recess matrix array, where N is an odd number. The biochip  100  is fabricated on 8′ wafers, and diced and assembled in PCBs with circuitry and adaptor connect to an external power supply. All odd electrode stripes  107  are connected to AC signal, and the rest even electrode stripes  107  are connected to the ground. AC electric field is supplied with an artificial function generator, Agilent 33250A. SiO 2  beads are used in this example (Sigma S5631) to illustrate the effect of frequency of the applied electric field to the active distribution of target entities. The beads are ranged from 0.5-10 um (80% between 1-5 um), and are suspended in DI H 2 O with conductivity of 5.5 uS/m. 30 ul of the beads solution is dispensed on the operation surface  108 A, covering the microelectrode array and the EK manipulation process is recorded via Nikon eclipse i50 microscope under white light. 
         [0109]    The images in  FIG. 9B  were collected to demonstrate the transportation and focusing effect of using patterned N by N matrix operation surface  108 A. The applied AC signal is 20Vpeak-to-peak sinewave, with frequency varying from 100 Hz to 1 MHz. 
         [0110]    When started at 100 Hz, beads started moving slowly. As frequency gradually increased to several kHz, the movement became more dramatic, and the centre of the well/recess  101  became more concentrated with beads. It was observed that, between 100 Hz to 500 Hz, the beads were drawn towards the centre of the electrode and the well/recess  101  and remained around the line of geometric symmetry. It was also observed that, as frequency increased above 500 Hz, the beads collected at the centre line started to be dragged into the nearest well/recess  101 . Consequently, the “line” split into “dots”. This was due to the profile described in  FIG. 4B . 
         [0111]    The most discrete round shape of beads cluster is observed when the frequency of the electric field is 10 kHz. As frequency increases beyond 10 kHz, the beads cluster started to deform and moves outside the well/recess  101  or the centre of the electrode  107  toward the edges of the electrode  107 . It was then observed that the beads were circulating from the edge of the electrode  107  towards the electrode centre, arising, and falling back to the edge region. The width of circulation became narrower as frequency increased. This process of transition was dramatic around 10 kHz to 20 kHz, and at 40 kHz, most beads were drawn into the furrows  104  and the gap of the electrodes  107 , as described in  FIG. 4D . 
         [0112]    From 40 kHz to 100 kHz, the width of circulation reaches minimum and the beads vibrate at electrode  107  edge, while some large sized beads exhibited self-rotation. Above 100 kHz, beads formed chains perpendicular to the electrode between the furrows  104 . The chains were then broken at around 600 kHz, and beads were repelled to the centre of the electrodes  107  and furrows  104 . This effect becomes more significant at 1 MHz. The reason why beads formed lines other than discrete dots as in low frequencies was because the dominating mechanisms of the fluid flow were different. More specifically, at low frequencies, beads experience ACEO induced fluid flow, which is sensitive to surface structures. The concave structure could regulate the flow and direct the beads inwards. While at high frequencies like in MHz level, ACEO no longer existed, ACET took dominance, which circulated beads from the bulk, and was less sensitive to surface structures. 
         [0113]    Referring to  FIG. 10 , there is shown the simulated DEP force field (red arrow) in a high conductivity solution medium with high frequency electric field. All arrows are directed away from the electrodes  107  and recesses  101 . 
         [0114]    Turning to  FIGS. 11A and 11B , in a low conductivity medium and with low frequency electric field, the DEP force, shown by arrows, is directed towards the well/recess  101  and the furrow  104  and away from the operation surface  108 A adjacent the well/recess  101 . The same applies when the solution medium is of low conductivity and a medium frequency electric field is applied (see  FIGS. 12A and 12B ) except that the local field is more concentrated towards the well/recess  101 . In a low conductivity medium and high frequency electric field as shown in  FIGS. 13A and 13B , the DEP force is directed away from the well/recess  101  and the furrow  104  but towards the operation surface  108 A adjacent the well/recess  101 . In  FIGS. 14A and 14B , when the conductivity of the medium is high and the frequency of the electric field is low, the DEP force directs away from the well/recess  101  and the furrow  104  but towards the operation surface  108 A adjacent the well/recess  101 . In  FIGS. 15A and 15B , when the solution medium is of high conductivity and the electric field is at high frequency, the DEP force are directed away from the well/recess  101  and the furrow  104  but towards the operation surface  108 A adjacent the well/recess  101 . 
         [0115]    In summary of a specific biochip design described above, in a high conductivity medium, even if the applied electric field has a low frequency, the DEP force is directed away from the well/recess  101 . To direct the DEP force towards the well/recess  101 , low conductivity medium should almost always be used. When directing the DEP force away from the well/recess  101 , a high frequency should be used and the conductivity of the medium is not of a major concern. This may not be a universal solution that only low to medium frequencies and low conductive medium can direct entities into the array of well/recess  103 . The design of electrode  107  and the well/recess  103  pattern are more crucial for modulating combinations of EK forces and therefore the target entities in or out of the well/recess  103 . The design of the electrode  107  and the well/recess  103  structures may include the material, scales or shapes thereof. 
         [0116]    The biochip  100  produces even better results by enhancing the EK assisted hybridization. As shown in  FIG. 2 , probe entities are attached via surface engineering to the operation surface  108 A by covalent bonds to a chemical matrix in a conventional manner. The aim of the biochip  100  according to the invention is to enhance interaction between the target entities in the solution medium and probe entities  109  on the operation surface  108 A by using Electrokinetic (EK) effects. A simulated comparison between passive hybridization and EK assisted hybridization is shown in  FIG. 5 .  FIG. 16  is a photographic comparison between two biochips, the left shows the result of EK assisted hybridization and the right shows the results of passive hybridization. Clearly EK assisted hybridization produces better results.  FIG. 18  is the artificial unit intensity representation of the hybridization result on the two biochips in  FIG. 16 . 
         [0117]    We now turn to the fabrication method of the biochip  100 . The preferred method is shown in  FIG. 6 . 
         [0118]    The process is based on a CMOS compatible microelectronic fabrication. On a silicon substrate  106 , a thickness of oxide layer  105  is produced by thermal oxidation, and a metal layer  107  is constructed via aluminum sputtering. The electrode arrays are formed by dry etch of the metal layer  107 . Following metallization process, silicon oxide (SiO 2 ) dielectric layer  108  is deposited via PECVD over the upper surface defined by the metal layer  107 . The array of wells/recesses  101  as well as the wire-bonding pad micro-indentations are patterned and opened by dry etching. Then, a second SiO 2  layer  108  with thickness is deposited over the first SiO 2  layer using PECVD. The chip fabrication completes with a final dry etch process for complete opening of the wire-bonding pads. Further process involves surface treatment of the biochip  100  for immobilization of probe entities  109  into each well/recess  101 . The biochip  100  is now ready for use. 
         [0119]    Referring to  FIG. 7  showing a process based on a CMOS compatible microelectronic fabrication. On a silicon substrate  106 , a thickness of oxide layer  105  is produced by thermal oxidation, and a metal layer  107  is constructed via aluminum sputtering. The electrode arrays  107  are formed by dry etching of this metal layer  107 . Another mask is applied for the well/recess  101  patterning, and etching follows, to create the well/recess  101  on metal electrodes  107 . After that, a silicon oxide (SiO 2 ) dielectric layer is deposited via PECVD over the upper surface of the metal layer  107 . The wire-bonding pad micro-indentations are patterned and opened by dry etching. 
         [0120]    Referring to  FIG. 8  which is again based on a CMOS compatible microelectronic fabrication. On a silicon substrate  106 , a thickness of oxide layer  105  is produced by thermal oxidation. We apply photo mask for patterning wells/recesses  101 , and perform dry etching on the thermal oxide layer  105 . After that the wells/recesses  101  are formed on the oxide layer  105 . A metal layer of is deposited on the upper surface of the oxide layer  105  and constructed via aluminum sputtering. The electrode arrays are formed by dry etching of this metal layer  107 . After that, a silicon oxide (SiO 2 ) dielectric layer is deposited via PECVD over the upper surface defined on the metal layer  107 . The wire-bonding pad micro-indentations are patterned and opened by dry etching. 
         [0121]    The invention has been given by way of example only, and various other modifications of and/or alterations to the described embodiment may be made by persons skilled in the art without departing from the scope of the invention as specified in the appended claims.

Technology Classification (CPC): 6