Patent Document

CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims the benefit of priority to U.S. Provisional Application Ser. No. 62/219,671 filed Sep. 17, 2015, titled “Dual-Sided Biomorphic Bioflex Polymer-based Microelectrode Array and Fabrication Thereof,” the contents of which are hereby incorporated by reference in their entirety. 
     
    
     FIELD 
       [0002]    The present invention relates generally to electrochemical recordings in biological systems and more specifically dual-sided biomorphic bioflex polymer-based microelectrode arrays and a method of fabrication of the same. 
       BACKGROUND OF THE INVENTION 
       [0003]    The detection of neurotransmitters and metabolic molecules requires reproducible and biologically inert microelectrode configurations using materials that will handle the high ionic strength and protein environments of central nervous system tissues. Historically, electrochemical recordings of neurotransmitters (including glutamate, acetylcholine, choline, GABA, adenosine, dopamine, norepinephrine, and serotonin) and metabolic molecules (including oxygen, glucose, lactate, pyruvate, and ATP) have focused on ways to minimize the “non-Faradaic” or non-specific background signals such as solvent dipole reorientation, adsorption, desorption, and movement of electrolyte ions at the recording site surfaces from measurements such that the major signal measured is due to the analyte of interest. In biological systems there are many contributors to changes in an electrode&#39;s background, or non-Faradaic response, such as pH shifts and changes in Ca 2+ , Na + , or Cl −  ions. 
         [0004]    Self-referencing has been an extremely powerful tool for removing the effects of chemical interferents that might contribute to a portion of the analyte signal and for permitting the subtraction of noise, which is present on both the analyte and control sites, from the analyte signal. One of the biggest advantages of self-referencing is that it makes it possible to measure both tonic levels and phasic changes of the neurochemical, rather than only the changes from a baseline or transient neurotransmitter changes. However, self-referencing subtraction cannot be achieved using single microelectrodes. 
         [0005]    Fabrication of microelectrodes using the basic concepts of photolithographic formation of microelectrodes on silicon substrates was used in the development of ceramic, substrate-based biomorphic electrode arrays (bMEAs), allowing for single-sided recording sites. However, while ceramic bMEAs have proven useful, there remain a number of issues surrounding the use of the bMEAs in biological systems such as their fragility, limitations of post lithographic cutting, the complexity of dual-sided configurations and the size limitations of ceramic substrates. 
         [0006]    It is within the aforementioned context that a need for the present invention has arisen. Thus, there is a need to address one or more of the foregoing disadvantages of conventional systems and methods, and the present invention meets this need. 
       BRIEF SUMMARY OF THE INVENTION 
       [0007]    Various aspects of dual-sided biomorphic polymer-based microelectrode arrays and fabrication thereof can be found in exemplary embodiments of the present invention. 
         [0008]    In a first embodiment, a biomorphic microelectrode array (bMEA) allows chemical and metabolic studies in multiple brain sub-regions simultaneously without moving the recording device up and down as well as the dual matching front and back recording sites for self-referencing recording described below [08]. Multiple recording sites allow for tonic and phasic recordings of neurochemical and metabolic molecules that cannot be measured by single microelectrodes. The multisite aspect of the sensor provides for examination of neurotransmitter system function at multiple brain locations and within brain structures. 
         [0009]    In another embodiment, microelectrodes are coated to have sentinel and analyte-sensing sites in close, defined proximity A dual-sided bMEA with identical recording sites on both the front and back of the microelectrode has one side coated with enzymes for detection of a specific analyte such as glutamate or glucose, lactate and a variety of other molecules, and the reverse side is coated as a control for self-referencing. Recording surfaces of matching platinum (Pt) crystal structure produce similar responses of catalyzed oxidation and/or reduction of reporter molecules enhancing the signal-to-noise of these microelectrodes for brain recordings. A front-back design allows for optimum self-referencing subtraction of non-Faradaic background signals, elimination of noise and subtraction of unknown chemical interferents. 
         [0010]    In a further embodiment, bMEAs are mass fabricated using photolithographic techniques and cut out using lithography allowing for the formation of a low insertion force tip unlike prior designs. Hundreds of bMEAs with different or identical designs can be simultaneously patterned on a single polymer wafer. High purity Pt (0.25 μm) is sputtered onto the recording sites. bMEAs with 4-8 recording sites have been fabricated with recording sites ranging from 10×10 μm to 50×300 μm. Various recording site sizes and arrangements can be selected to conform to specific brain layers. The front and back of the polymer substrate are patterned to increase recording site density and create isolated front and back site recording pairs, where if one site or pair fails useful data can be received from the other recording sites. 
         [0011]    A further understanding of the nature and advantages of the present invention herein may be realized by reference to the remaining portions of the specification and the attached drawings. Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with respect to the accompanying drawings. In the drawings, the same reference numbers indicate identical or functionally similar elements. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1A  illustrates a microelectrode probe system according to an exemplary embodiment of the present invention. 
           [0013]      FIG. 1B  illustrates a microelectrode site cross section within an exemplary probe system according to an exemplary embodiment of the present invention. 
           [0014]      FIG. 1C  illustrates a biomorphic multi-site microelectrode array within an exemplary probe system according to an exemplary embodiment of the present invention. 
           [0015]      FIG. 2  illustrates a profile of a dual-sided biomorphic polymer-based microelectrode array in accordance with an exemplary embodiment of the present invention. 
           [0016]      FIG. 3  illustrates enzyme-based coatings of microelectrodes in accordance with an exemplary embodiment of the present invention. 
           [0017]      FIG. 4  illustrates a process for fabricating dual-sided biomorphic polymer-based microelectrode arrays in accordance with an exemplary embodiment of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0018]    Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention as defined by the appended claims. Furthermore, in the following detailed description of the present invention, numerous specific details are set forth to provide a thorough understanding of the present invention. However, it will be obvious to one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail as to not unnecessarily obscure aspects of the present invention. 
         [0019]      FIG. 1A  illustrates a microelectrode probe system  100  according to an exemplary embodiment of the present invention. 
         [0020]    In  FIG. 1A , microelectrode probe system  100  comprises, among other components, interface  101  containing conductive contacts  102  connected with communication channels  104  and other contacts  103 . Interface  101  is physically coupled with probe body  105  comprising housing  106  and tip  107 , both containing other probe components. Communication channels  104  can be any medium that allows communication from one point to another, for example for transmitting signals from components within probe body  105  to conductive contacts  102 . As shown, microelectrode probe system  100 , probe body  105 , housing  106 , and tip  107  may be composed of a single physical layer or a combination of a plurality of physical layers. 
         [0021]    Probe body  105 , housing  106 , and tip  107  may be rigid or flexible. Microelectrode probe system  100  is used to measure chemical and electrical values using components within probe housing  106  and probe tip  107 . Although not shown, other devices may be connected to microelectrode probe through interface  101  and conductive contacts  102  to retrieve information from probe components through communication channels  104 . Components within the probe housing  106  and probe tip  107  can include any type of sensor or network of sensors appropriate for the desired physiological measurements to be collected. 
         [0022]      FIG. 1B  illustrates a microelectrode site cross section  110  within a probe system according to an exemplary embodiment of the present invention. It will be appreciated that microelectrode site cross section  110  as depicted in  FIG. 1B  is illustrative of a cross section of an exemplary probe system, similar to the example provided by  FIG. 1A . 
         [0023]    In  FIG. 1B , microelectrode site cross section  110  within an exemplary probe system comprises probe body  105  and housing  106  containing communication channels  104  individually coupled with a microelectrode  111 . It will be appreciated that communication channels  104  within housing  106  can be communicably coupled with communication channels  104  within interface  101  as depicted in  FIG. 1A . Probe body  105  and housing  106  may be rigid or flexible. Communication channels  104  can be any medium that allows communication from one point to another for example for transmitting signals from microelectrode  111  within probe body  105  to conductive contacts  102  within interface  101  as depicted in  FIG. 1A . Microelectrode  111  performs measurement of characteristics of a substance and communicates measurement information through communication channel  104 . As shown, microelectrode site cross section  110  within an exemplary probe system, probe body  105 , and housing  106  may be composed of a single layer or a combination of a plurality of physical layers. 
         [0024]      FIG. 1C  illustrates a biomorphic multi-site microelectrode array within a probe system  120  according to an exemplary embodiment of the present invention. 
         [0025]    In  FIG. 1C , biomorphic multi-site microelectrode array within an exemplary probe system  120  comprises, among other things, probe body  105 , housing  106 , and tip  107 . Probe body  105 , housing  106 , and tip  107  may be rigid or flexible. Housing  106  and tip  107  contain a plurality of microelectrodes  111  each coupled with an individual communication channel  104 . 
         [0026]    Microelectrode  111  may be placed at variable locations within housing  106  in order to achieve specific measurements at a given probe location. Biomorphic multi-site microelectrode array within a probe system  120  contains a plurality of microelectrodes  111  of no fixed amount. Placement and spacing of microelectrodes  111  in a linear and/or paired arrangement (100 microns to 3 mm) is designed to match a variety of brain structures affording a biomorphic array allowing simultaneous recordings at multiple target depths and coordinates from one microelectrode probe system  100 . 
         [0027]      FIG. 2  illustrates a profile of a dual-sided biomorphic polymer-based microelectrode array  200  according to an exemplary embodiment of the present invention. 
         [0028]    In  FIG. 2 , dual-sided biomorphic polymer-based microelectrode array  200  comprises an upper side  200 A and a lower side  200 B, the upper and lower sides separated by polymer and/or other material layer  201 , each side containing interface  101  physically coupled with flexible probe body  105  comprising housing  106  and tip  107 . Housing  106  and tip  107  contain a plurality of microelectrodes (not shown, although examples are depicted in the preceding Figures, for example microelectrode  111 ) each connected to an individual communication channel (not shown, although examples are depicted in the preceding Figures, for example communication channel  104 ). It will be appreciated that, while probe body  105  may appear to be connected to and not containing house  106  and tip  107  in the Figures herein, probe body  105  comprises both house  106  and tip  107 . 
         [0029]    According to one embodiment, an individual microelectrode (not shown)  111  on one side of dual-sided biomorphic polymer-based microelectrode array  200  is responsible for measurement of a specific analyte such as glutamate or glucose, lactate and a variety of other molecules. Each microelectrode  111  is paired with a corresponding microelectrode  111  on the reverse side of dual-sided biomorphic polymer-based microelectrode array  200 . The corresponding microelectrode  111  is responsible for detection of a specific analyte and is used as a control for self-referencing. Enzyme-based coatings on microelectrodes  111  on one side specific to analytes of interest (active), coupled with a similar but non-functional protein matrix (sentinel) coating on the microelectrode  111  on the opposite side yields two distinct recording sites for subtraction of interferents, noise and non-Faradaic background current. Differences in the measurement recorded on paired microelectrodes  111  on each side of the dual-sided biomorphic polymer-based microelectrode array  200  provide for the identification and elimination of interfering signals. Dual-sided biomorphic polymer-based microelectrode array  200  allows for improved signal to noise affording tonic and phasic recordings of neurochemical and metabolic molecules that cannot be measured by single microelectrodes. The disclosures of Burmeister and Gerhardt, Analytical Chem 2001, 73 and Miller et al, J Neuroscuience Methods, 252 (2015) provide explanation of self-referencing and improved signal to noise as referenced herein; the disclosures of both publications are hereby incorporated by reference in their entirety. 
         [0030]      FIG. 3  illustrates enzyme-based coatings of microelectrodes  300  in accordance with an exemplary embodiment of the present invention. 
         [0031]    In  FIG. 3 , enzyme-based coatings of microelectrodes  300  comprise three layers. Communication layer  301  is separated from enzyme layer  303  by a barrier layer  302 . Communication layer  301  may be comprised of a sputtered platinum (Pt) metal layer to produce catalyzed oxidation of molecules (or reduction depending on analyte measured)  306  for reporting measurements. Barrier layer  302  may be comprised of poly-(meta-phenylenediamine) (mPD) to minimize the oxidization of unwanted molecules applied to the communication layer  301 . Enzyme layer  303  comprises an enzyme coating specific or highly selective to an analyte to be measured. Enzyme layer  303  is also referred to as an enzyme coating on the recording site. 
         [0032]    When the enzyme layer  303  is coated with a specific enzyme such as, but not limited to, glutamate oxidase, allowed molecules  304  such as glutamate interact and pass  305  reporter molecules such as but not limited to H 2 O 2    306  to the communication layer  301 . Other molecules  307  cannot either interact with the selective enzyme layer and/or are prevented from passing  308  certain oxidizable and/or reducible molecules to the communication layer  301  by the barrier layer  302 . 
         [0033]      FIG. 4  illustrates a process  400  for fabricating dual-sided biomorphic polymer-based microelectrode arrays in accordance with an exemplary embodiment of the present invention. 
         [0034]    In  FIG. 4 , a microfabrication process  400  starts with flexible polymer wafer  401  such as laminated Kapton blank polymer wafer or acrylic substrate. Lift-off layer (LOL) is then spun  402  onto the wafer and pre-baked. 
         [0035]    In a next lithography step  403 , the LOL is exposed to light in a mask aligner and etched to open areas for TiPt metal deposition. 
         [0036]    In a metal coating step  404 , high purity Pt (0.25 μm) is sputtered conformally. The Pt on LOL is then lifted and removed  405 , and Pt on polymer substrate is left on the polymer wafer surface forming the desired device pattern. 
         [0037]    Next the wafer undergoes a polyimide coating and lithography step  406 . The wafer with Pt pattern is conformally spin-coated with polyimide layer (0.2 μm), and pre-baked. The wafer is then coated with photo resist (PR), baked, and exposed to the light in a mask aligner. 
         [0038]    Finally, the wafer is etched  407  in a developer to open areas for etching polyimide and finish formation of electrical insulating coating on conducting traces. The etching defines precisely the areas of recording sites of the electrodes. 
         [0039]    According to one embodiment, the process  400  described in  FIG. 4  includes a high definition lift-off process for thicker Ti/Pt deposition (e.g., greater than 0.16 um). The lift-off process is optimized to minimize Ti/Pt lifting at the contour of breaking through the metal. A thicker Ti/Pt layer enables reliable wire bonding and soldering of electrical connectors. This provides more options in electrical connector selection and packaging types. 
         [0040]    According to one embodiment, sputtering is used in the process  400  described in  FIG. 4  for the benefit of lower temperatures as compared to those when e-beam evaporation is used, and the sputtering is done in stages for controlling temperatures below 90 degrees Celsius on the substrate. 
         [0041]    According to one embodiment, the process  400  described in  FIG. 4  is applicable to both rigid substrates (alumina—Al2O3) and flexible substrates (Kapton—polyimide, acrylic, etc.). Rigid substrates are more limited in size as compared to flexible substrates which can be substantially larger in size. Flexible substrates and rigid substrates differ in packaging in that, in the case of flexible substrates, it can be easier to attach connectors and/or create connectors by soldering pins to the substrate. 
         [0042]    The process  400  described in  FIG. 4  can also be used on other types of substrates such as acrylic (PMMA), silicon (Si) and the like. 
         [0043]    According to one embodiment, thin polyimide and/or SU-8 conformal coating applied over the conducting traces serves for electric insulation purposes and in some designs to define geometry of electric recording sites. Long (on the scale of 100 mm or longer) and thin (less than 0.25 mm×0.25 mm cross section) polyimide flexible probes minimize damage and inflammation in brain tissue. 
         [0044]    According to one embodiment, a stainless steel wafer is used in process  400 . The polymer layer(s) are then be layered on each side of the stainless steel wafer, enabling creation of an easier to fabricate, more resilient and thinner probe. This design also provides an within probe ground plane, electrical stimulation and pseudo-reference electrode. It will be appreciated that the use of a stainless steel wafer is a complement for dealing with optogenetics. 
         [0045]    According to one embodiment, channel multiplexing is used to serialize a potentially large number of communication channels. Such a design can reduce the number of pins or lines required for the interface. Such channel multiplexing can be achieved using semiconductor chips. It will be appreciated that channel multiplexing can be utilized in a design employing a stainless steel wafer or other wafer materials disclosed herein without departing from the scope of the present disclosure. 
         [0046]    Table 1 below lists possible enzymes used to create the enzyme reactive layer and associated selective analyte measurement. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Enzyme - Analyte Measurement Combinations. 
               
             
          
           
               
                 Analyte Measured 
                 Enzyme 
                 Substrate 
                 Product 
               
               
                   
               
               
                 Glutamate 
                 Glutamate oxidase 
                 Glutamate, O 2   
                 H 2 O 2 , α-ketoglutarate 
               
               
                 Choline 
                 Choline oxidase 
                 Choline, O 2   
                 H 2 O 2 , Betaine 
               
               
                 Lactate 
                 Lactate oxidase 
                 Lactate, O 2   
                 H 2 O 2 , Pyruvate 
               
               
                 Glucose 
                 Glucose oxidase 
                 Glucose, O 2   
                 H 2 O 2 , gluconic acid 
               
               
                 Acetylcholine 
                 1. Acetylcholinesterase 
                 1. Acetylcholine  
                 1. Choline, Acetic Acid 
               
               
                   
                 2. Choline Oxidase 
                 2. Choline, O 2   
                 2. H 2 O 2 , Betaine 
               
               
                 Remove H 2 O 2  or  
                 Catalase  
                 H 2 O 2   
                 O 2   
               
               
                 used as a mediator 
                   
                   
                   
               
               
                 ATP 
                 1. glycerol kinase 
                 1. Glycerol, ATP 
                 1. Glycerol-3- 
               
               
                   
                 2. Glycerol-3-phospate 
                 2. glycerol-3- 
                 phosphate, ADP 
               
               
                   
                 oxidase 
                 phosphate, O 2   
                 2. H 2 O 2 , 
               
               
                   
                   
                   
                 dihydroxyacetone 
               
               
                   
                   
                   
                 phosphate 
               
               
                 GABA 
                 1. Gabase 
                 1. GABA, NADP + ,  
                 1. Glutamate, 
               
               
                   
                 2. Glutamate Oxidase 
                 α-ketoglutarate 
                 NADPH, H +   
               
               
                   
                   
                 2. Glutamate, O 2   
                 2. H 2 O 2 , 
               
               
                   
                   
                   
                 α-ketoglutarate 
               
               
                 Pyruvate 
                 Pyruvate oxidase 
                 Pyruvate, O 2 , 
                 acetyl phosphate, 
               
               
                   
                   
                 phosphate 
                 H 2 O 2 , CO 2   
               
               
                 Xanthine 
                 Xanthine oxidase 
                 Xanthine, O 2   
                 H 2 O 2 , Uric acid, 
               
               
                 Inosine 
                 1. Nucleoside 
                 1. Inosine, 
                 1. ribose-1-phosphate, 
               
               
                   
                 phosphorylase 
                 phosphate 
                 hypoxanthine 
               
               
                   
                 2. Xanthine oxidase 
                 2. Xanthine, O 2   
                 2. H 2 O 2 , Uric acid 
               
               
                 Adenosine 
                 1. Adenosine deaminase 
                 1. Adenosine, H 2 O 
                 1. Inosine, NH 3   
               
               
                   
                 2. Nucleoside  
                 2. Inosine, 
                 2. ribose-1-phosphate, 
               
               
                   
                 phosphorylase 
                 phosphate 
                 hypoxanthine 
               
               
                   
                 3. Xanthine oxidase 
                 3. Xanthine, O 2   
                 3. H 2 O 2 , Uric acid 
               
               
                 Alcohols 
                 Alcohol oxidase 
                 alcohol, O 2   
                 H 2 O 2 , aldehyde 
               
               
                 D-amino acids 
                 D-amino acid oxidase 
                 D-amino acid, O 2   
                 H 2 O 2 , 2-oxo acid, NH 4   +   
               
               
                 L-amino acids 
                 L-amino acid oxidase 
                 L-amino acids, O 2   
                 H 2 O 2 , 2-oxo acid, NH 4   +   
               
               
                 Ascorbate Removal 
                 Ascorbate oxidase 
                 Ascorbate, O 2   
                 Dehydroascorbate 
               
               
                 Aspartate 
                 Aspartate oxidase 
                 Aspartate, O 2   
                 H 2 O 2 , NH 4   +   
               
               
                   
                   
                   
                 Oxaloacetate, 
               
               
                 Cholesterol Ester 
                 1. Cholesterol esterase 
                 1. Cholesteryl 
                 1. Cholesterol, oleic 
               
               
                   
                 2. Cholesterol oxidase 
                 oleate or other 
                 acid or other 
               
               
                   
                   
                 Cholesterol ester, 
                 corresponding ketone 
               
               
                   
                   
                 taurocholate 
                 2. H 2 O 2 , 4-cholesten-3- 
               
               
                   
                   
                 2. Cholesterol, O 2   
                 one 
               
               
                 Cholesterol  
                 Cholesterol oxidase 
                 Cholesterol, O 2   
                 H 2 O 2 , 4-cholesten-3- 
               
               
                   
                   
                   
                 one 
               
               
                 D-Serine 
                 D-amino acid oxidase 
                 D-Serine, O 2   
                 H 2 O 2 , corresponding 
               
               
                   
                 (DAAO) 
                   
                 imino acids, NH 4   +   
               
               
                 Galactose 
                 Galactose oxidase 
                 D-galactose, O 2   
                 H 2 O 2 , D- 
               
               
                   
                   
                   
                 galactohexodialose 
               
               
                 Glutamine 
                 1. Glutaminase 
                 1. Glutamine 
                 1. Glutamate, NH 4 + 
               
               
                   
                 2. Glutamate Oxidase 
                 2. Glutamate, O 2   
                 2. H 2 O 2 , α- 
               
               
                   
                   
                   
                 ketoglutarate 
               
               
                 Mediator for H 2 O 2   
                 Horseradish peroxidase 
                 H 2 O 2   
                 H 2 O, O 2   
               
               
                 detection 
                   
                   
                   
               
               
                 Lysine 
                 Lysine oxidase 
                 Lysine, O 2   
                 H 2 O 2 , NH 4   + , 
               
               
                   
                   
                   
                 6-amino-2-oxohexanoic 
               
               
                   
                   
                   
                 acid 
               
               
                 Sarcosine 
                 Sarcosine oxidase 
                 Sarcosine, O 2   
                 H 2 O 2 , Glycine, 
               
               
                   
                   
                   
                 Formaldehyde 
               
               
                   
               
             
          
         
       
     
         [0047]    Polyimide flexible probes with simultaneous multiple recording sites detecting signals from individual neurons have been disclosed herein. Double sided flexible brain probes are built herein on a flexible wafer using all biocompatible materials and processes: polyimide—Kapton, acrylic—SU8, conformal coatings, Pt metallization, lift-off process. 
         [0048]    While the above is a complete description of exemplary specific embodiments of the invention, additional embodiments are also possible. Thus, the above description should not be taken as limiting the scope of the invention, which is defined by the appended claims along with their full scope of equivalents.

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