[CONFIDENTIAL TREATMENT REQUESTED. CONFIDENTIAL PORTIONS OF THIS AGREEMENT HAVE BEEN REDACTED AND HAVE BEEN SEPARATELY FILED WITH THE COMMISSION.] Exhibit 10.11 FIRST AMENDMENT TO THE AGREEMENT ENTITLED "ASSIGNMENT OF PATENT RIGHTS AND TECHNOLOGY" ENTERED INTO AUGUST 1, 1991 BETWEEN THE UNIVERSITY OF TEXAS, ADAM HELLER, AND E. HELLER AND COMPANY (THE "AMENDMENT") WHEREAS, under the August 1, 1991, agreement entitled "Assignment of Patent Rights and Technology" (hereafter "Assignment"), and Exhibit A hereto, the Board of Regents of the University of Texas System on behalf of The University of Texas at Austin (hereafter collectively referred to as "Assignor") assigned to E. Heller and Company ("Assignee") its right, title, and interest in, to, and under the Patent Rights and the Technology (as such terms are defined in the Assignment); and WHEREAS, under Paragraph 3.1 of the Assignment the parties agreed that in return for its assignment of rights, Assignor would be compensated as noted in the event Patent Rights, the Technology, and the Know-How are sold, licensed, or otherwise disposed of for value to a third party or parties; and under Paragraph 3.2 of the Assignment the parties also agreed that Assignor would be compensated for a to-be-negotiated amount should Assignee elect to itself manufacture, sell, or otherwise dispose of for value Licensed Products and Licensed Processes (as such terms are defined in the Assignment) and which are derived from the Patent Rights, the Technology, and the Know-How; and WHEREAS, since the effective date of the Assignment, Assignee has invested approximately *** in research and development of biosensors within the scope of the Technology, Patent Rights, and Know-How, and estimates that an additional investment of approximately *** will be necessary to complete product development and enter into clinical trials, and Assignee, on behalf of itself and its Affiliates (as defined below), has now elected to manufacture, sell, or otherwise dispose of for value, including sublicensing, the Patent Rights, Technology, Know-How, Licensed Products, and Licensed Processes; and WHEREAS, Assignor and Assignee now desire to amend Paragraphs 3.1 and 3.2 and certain other paragraphs of the Assignment and specifically set out herein the mutually agreed compensation due Assignor for Assignee's disposition for value of the Patent Rights, the Technology, the Know-How, Licensed Products and Licensed Processes. NOW, THEREFORE, in consideration of the covenants and agreements herein contained the parties agree as follows: 1. All capitalized terms not defined in this Amendment shall have the meaning given to them in the Assignment. 2. Section 1.2 is amended to read in its entirety as follows: 1.2 Patent Rights. The term "Patent Rights" as used herein shall mean ------------- Assignor's rights in all inventions and intellectual property disclosed in the patent applications patents and other invention disclosures listed on Exhibit B and Exhibit C hereto, including all U.S. and *** Confidential treatment requested foreign patent applications including provisional applications, and all divisions, continuations, continuations in-part, and substitutions thereof; all foreign patent applications corresponding to the preceding applications; and all U.S. and foreign patents issuing on any of the preceding applications, including extensions, reissues, and re- examinations. 3. Section 1.3 is amended to read in its entirety as follows: 1.3 Know-How. The term "Know How" as used herein shall mean -------- Assignor's rights in all ideas, inventions, data, trade secrets, instructions, processes, formulas, chemical, pharmacological, toxicological, pharmaceutical, physical and analytical, manufacturing (including but not limited to processes, yields, reagants and conditions relating to compound manufacture) data and information, owned or controlled by Assignor existing as of the last signature date of this Assignment which are generally not known, and which are necessary or useful for the manufacture of Licensed Products. Excluded from Know-How are any inventions included with Patent Rights. 4. Section 1.4 is amended to read in its entirety as follows: 1.4 Licensed Products. The term "Licensed Products" as used herein ----------------- shall mean any product which (i) is covered by a Valid Claim in the country such product is made or sold, or (ii) incorporates in material part or is made using the Technology, Know-How, Licensed Processes or Other Intellectual Property. For the avoidance of doubt, subject to the foregoing, a Licensed Product unit shall mean any instrument or associated disposable. 5. Article 1 is amended to add new Section 1.7 as follows: 1.7 Affiliate. The term "Affiliate" as used herein shall mean any --------- corporation or other entity which is directly or indirectly controlling, controlled by or under the common control with a party hereto. For the purposes of this definition, "control" shall mean the direct or indirect ownership of at least fifty percent (50%) of the outstanding shares or other voting rights of the subject entity to elect directors, or if not meeting the preceding, any entity owned or controlled by or owning or controlling at the maximum control or ownership right permitted in the country where such entity exists. 6. Article 1 is amended to add new Section 1.8 as follows: 1.8 Net Sales. The term "Net Sales" as used herein shall mean the --------- gross revenues received by Assignee, its Affiliates or sublicensees from sales of Licensed Products, less (i) normal and customary rebates, and cash and trade discounts, actually taken, (ii) sales, value-added, use and/or other excise taxes or duties actually paid, (iii) outbound transportation charges prepaid or allowed, (iv) import and/or export duties actually paid, and (v) amounts allowed or credited due to returns. 7. Article 1 is amended to add new Section 1.9 as follows: 1.9 Valid Claim. The term "Valid Claim" as used herein shall mean (i) ----------- a claim of an issued and unexpired patent included within the Patent Rights which has not been disclaimed -2- or admitted to be invalid or unenforceable through reissue or otherwise, or (ii) a claim of a pending application within the Patent Rights provided that not more than five (5) years have elapsed from the date the claim takes priority for filing purposes. 8. Article 1 is amended to add new Section 1.10 as follows: 1.10 Dominating Patent. The term "Dominating Patent" as used herein ----------------- shall mean an unexpired patent which is owned by a third party covering Licensed Products made and sold by Assignee or its sublicensees under circumstances such that Assignee or its sublicensee has no commercially reasonable alternative but to obtain a license under such patent in order to make, use or commercialize a Licensed Product. 9. Article 1 is amended to add new Section 1.11 as follows: 1.11 Other Intellectual Property. The term "Other Intellectual --------------------------- Property" as used herein shall mean Board's rights in any biosensor related inventions (whether or not patentable), improvements, discoveries, developments, original works of authorship, software, trade secrets, Know-How made, conceived, reduced to practice or otherwise developed, by an employee of Assignor pursuant to either (i) a consulting agreement entered into by such employee and EHC or its sublicensee; or (ii) any similar agreement between an employee of the University of Texas at Austin and Assignee, and all intellectual property rights therein and thereto, shall be subject to the Assignment in Section 2.1 below. 10. Section 2.1 is amended to read in its entirety as follows: 2.1 Assignment by Assignor. Assignor hereby transfers, assigns, ---------------------- sells, and conveys to Assignee, its successors and assigns, the entire right, title and interest throughout the world in and to the Patent Rights, Technology, Know-How, and Other Intellectual Property subject only to: (a) any rights of the United States Government which may exist now or in the future due to a research funding agreement to which the United States Government may be a party; and (b) the terms and conditions of this Agreement. 11. Article 3 is amended to read in its entirety as follows: 3.1 Royalties. In consideration for the Assignment herein, Assignee --------- shall pay to Assignor the greater of (i) an annual minimum royalty of twenty thousand dollars or (ii) royalties on Net Sales of Licensed Products sold by Assignee, an Affiliate or its sublicensees which are within the scope of a Valid Claim as follows: Numbers of Licensed Royalty Rate Products Sold Worldwide During the Term ------------ --------------------------------------- *** of Net Sales 1-100,000 *** of Net Sales 100,001-200,000 *** of Net Sales 200,001-and above *** Confidential treatment requested -3- Any minimum annual royalty payable hereunder shall be fully creditable against any running royalty payable to Assignor during such year. 3.2 Sublicense Payment. In addition to the royalties subject to ------------------ Section 3.1 above, Assignee shall pay to Assignor *** received by Assignee and its Affiliates from licensees and sublicensees of the Patent Rights, Know-How, Technology or Other Intellectual Property. Notwithstanding the foregoing, it is understood and agreed that Assignor shall not be entitled to any portion of amounts received from licensees or sublicensees for equity in Assignee less than *** of fair market value, debt financing, research and development funding, the license or sublicense of any intellectual property other than the Patent Rights, Know-how, Technology or Other Intellectual Property, or reimbursement for patent or other expenses, and to be determined in a manner consistent with generally accepted accounting principles (GAAP). 3.3 Royalties on Combination Products. In the event that a Licensed --------------------------------- Product is sold by Assignee, an Affiliate or its sublicensee in combination as a single product with another product whose sale and use are not within the scope of a Valid Claim in the country for which the combination product is sold, Net Sales from such sales for purposes of calculating the amounts due under Section 3.1 above shall be calculated by multiplying the Net Sales of that combination by the fraction A/(A + B), where A is the gross selling price of the Licensed Product sold separately and B is the gross selling price of the other product sold separately. In the event that no such separate sales are made by Assignee, an Affiliate or its sublicensee, Net Sales for royalty determination shall be as reasonably allocated by Assignee, an Affiliate or its sublicensee between such Licensed Product and such other product, based upon their relative importance and proprietary protection. Notwithstanding the above, in no event shall Assignor receive a royalty of less *** of the running royalty amount per unit of Licensed Product due under Section 3.1 above. 3.4 Third Party Royalty Offset. In the event Assignee, an Affiliate -------------------------- or its sublicensee enters into a license agreement with any third party with respect to a Dominating Patent or to settle a claim of infringement or misappropriation of any intellectual property of a third party relating to the practice or use of the Technology and or Know-How, Assignee, an Affiliate or its sublicensee may offset any payments made in accordance with such license agreements against any amounts of running royalties owned by Assignee pursuant to Article 3 herein up to a maximum of *** of the amount otherwise due to such third party; provided, however that in no event will the running royalties due to Assignor be lower than the following: Royalty Rate Number of Licensed Products Sold Worldwide ------------ ------------------------------------------ During the Term --------------- *** of Net Sales 1-100,000 *** of Net Sales 100,001-200,000 *** of Net Sales 200,001 - and above -4- *** Confidential treatment requested In addition, in such event, the amounts due to Assignor under Section 3.2 above shall be reduced by an amount equal to *** the amount paid to such third party. 3.5 One Royalty. No more than one royalty payment shall be due with ----------- respect to a sale of a particular Licensed Product. No multiple royalties shall be payable because any Licensed Product, or its manufacture, use or sale is covered by more than one Valid Claim. No royalty shall be payable under this Article 3 with respect to Licensed Products distributed for use in research and/or development, in clinical trials or as promotional samples. 3.6 Royalty Term. Royalties due under this Article 3 shall be payable ------------ on a country-by-country and Licensed Product-by-Licensed Product basis until the expiration of the last-to-expire issued Valid Claim covering such Licensed Product in such country, or if no such Valid Claim issued in a country, until the fifth anniversary of the first commercial sale of a Licensed Product in such country. 3.7 In the event Assignee engages in litigation and/or settlement negotiations to advance a claim it makes that a third party is infringing on Licensed Products and/or Licensed Processes, *** on any litigation or settlement recovery by Assignee will be paid to Assignor; provided, however, that Assignee shall be entitled to first deduct reasonable and documented attorney's and other professional fees, expert fees, court costs, and other reasonable expenses incurred by Assignee in such litigation and/or settlement activities. 12. Section 4.2 is amended to read in its entirety as follows: 4.2 Payments and Reports. The first annual minimum royalty payment of -------------------- twenty thousand dollars ($20,000) shall be due and payable to Assignor within 30 days of the date of the last signature on this Amendment. Thereafter, minimum annual royalty payments, when due, shall be due and payable within thirty (30) days after the end of the applicable year. All running royalties payable hereunder by Assignee shall be due within thirty (30) days following the end of each respective semi-annual reporting period, which ending dates shall be June 30 and December 31 for each successive year. Each report will accurately identify income and expenses in sufficient form and detail so as to enable Assignor to determine the royalties due for such semi-annual period, and shall be mailed to the following: Office of the Executive Vice President and Provost, The University of Texas at Austin, Main Building 201, Austin, Texas 78712-1111, ATTN: Patricia C. Ohlendorf, with a copy to the Office of Technology Licensing and Intellectual Property at the address listed in 16.3 herein. Checks shall be made payable to The University of Texas at Austin. Except as expressly provided herein, all amounts payable hereunder shall be payable in United States dollars without deductions for taxes, assessments, fees, or charges of any kind. 13. Article 15 is amended to read in its entirety as follows: 15.1 Assignment. Neither party may assign this Assignment without the ---------- prior consent of the other, which consent shall not be unreasonably withheld; provided, however, Assignee may assign this Assignment in connection with a transfer of all or substantially all of its *** Confidential treatment requested -5- assets relating to this Assignment, whether by sale, merger, operation of law or otherwise. This Assignment shall be binding upon and inure to the benefit of the parties and their successors and assigns. 14. New Article 16 is added as follows: MISCELLANEOUS ------------- 16.1 Affiliate Rights/Obligations. Assignee shall have the right to extend ---------------------------- the provisions of the Assignment regarding the right to manufacture, sell, or otherwise dispose of for value, including licensing and sublicensing, the Patent Rights, Technology, Know-How, Other Intellectual Property, Licensed Products, and Licensed Processes, to any Affiliate, provided such Affiliate consents to be bound by the Assignment to the same extent as Assignee. Further, Assignee shall have the right to extend the provisions of this Amendment in their entirety to any Affiliate, provided such Affiliate consents to be bound by the applicable provisions of this Amendment to the same extent as Assignee. 16.2 Entire Agreement. The Assignment and this Amendment constitute the ---------------- entire and only agreement between the parties with respect to the Patent Rights, Technology, Know-How, Other Intellectual Property, Licensed Products, and Licensed Processes, and all other prior negotiations, representations, agreements, and understandings are superseded hereby. No agreement altering or supplementing the terms of the Assignment may be made except by means of a written document signed by a duly authorized representative of each party. 16.3. Notices. Any notices under this Assignment or shall be given by ------- prepaid, first class, certified mail, return receipt requested, addressed as follows (or such other address as may be given from time to time under the terms of this provision): in the case of Assignor - with a copy to - BOARD OF REGENTS OFFICE OF THE EXECUTIVE The University of Texas System VICE PRESIDENT & PROVOST 201 West 7th Street The University of Texas at Austin Austin, Texas 78701 Austin, Texas 78712-1111 ATTN: System Intellectual Property Office ATTN: Patricia C. Ohlendorf with a copy to - OFFICE OF TECHNOLOGY LICENSING AND INTELLECTUAL PROPERTY The University of Texas at Austin MCC Building Suite 1.9A 3925 West Braker Lane Austin, Texas 78759 ATTN: Director -6- or in the case of Assignee - E. HELLER AND COMPANY 1311 Harbor Bay Parkway, Suite 1000 Alameda, California 94502 ATTN: Ephraim Heller 16.4 LIMITATION OF LIABILITY. SUBJECT TO ARTICLE XIV OF THE ----------------------- ASSIGNMENT, NEITHER PARTY SHALL BE LIABLE TO THE OTHER FOR ANY SPECIAL, CONSEQUENTIAL, INCIDENTAL OR INDIRECT DAMAGES ARISING OUT OF THIS ASSIGNMENT, HOWEVER CAUSED, UNDER ANY THEORY OF LIABILITY 16.5 Right to Independently Develop. Nothing in this Assignment will ------------------------------ impair Assignee's right to independently acquire, license, develop for itself, or have others develop for it, technology or intellectual property performing the same or similar functions as the Know-How or the Patent Rights, or the Other Intellectual Property, or to market and distribute licensed products based on such other intellectual property and technology. 16.6 Further Assurances. At any time or from time to time on and after ------------------ the date of this Assignment, Assignor shall at the request and sole expense of Assignee (i) deliver to Assignee such records, data or other documents consistent with the provisions of this Assignment, (ii) execute, and deliver or cause to be delivered, all such consents, documents or further instruments of transfer or license, and (iii) take or cause to be taken all such actions, as Assignee may reasonably deem necessary or desirable in order for Assignee to obtain the full benefits of the Assignment and Amendment and the transactions contemplated hereby. 16.7 Severability. In the event that any provisions of this Assignment ------------ are determined to be invalid or unenforceable by a court of competent jurisdiction, the remainder of the Assignment shall remain in full force and effect without said provision. The parties shall in good faith negotiate a substitute clause for any provision declared invalid or unenforceable which shall most nearly approximate the intent of the parties in entering this Assignment. 16.8 Modification; Waiver. This Assignment may not be altered, amended -------------------- or modified in any way except by a writing signed by both parties. The failure of a party to enforce any provision of the Assignment shall not be construed to be a waiver of the right of such party to thereafter enforce that provision or any other provision or right. 15. Except as specifically modified or amended hereby, the Assignment shall remain in full force and effect and, as so modified or amended, is hereby ratified, confirmed and approved. No provision of this Amendment may be modified or amended except expressly in a writing signed by both parties nor shall any terms be waived except expressly in a writing signed by the party charged therewith. This Amendment shall be governed in accordance with the laws of the State of Texas, without regard to principles of conflicts of laws. -7- IN WITNESS WHEREOF, this Amendment effective as of March 19, 1998, is hereby executed by the duly authorized representatives of the parties on the date indicated below in duplicate, each of which shall be deemed an original and together shall form one and the same instrument. BOARD OF REGENTS OF THE UNIVERSITY OF TEXAS SYSTEM E. HELLER AND COMPANY /s/ Ray Farabee /s/ Ephraim Heller --------------------------------------- ------------------------------- Ray Farabee Ephraim Heller Vice Chancellor and General Counsel President Date: 4/7/98 Date: 3/19/98 ---------------------------------- -------------------------- APPROVED AS TO CONTENT ADAM HELLER /s/ Patricia C. Ohlendorf /s/ Adam Heller --------------------------------------- ------------------------------- Patricia C. Ohlendorf Adam Heller Counsel to the President Date: 3/19/98 -------------------------- Vice Provost Date: 3/27/98 ---------------------------------- APPROVED AS TO FORM /s/ Georgia Harper --------------------------------------- Name: Georgia Harper ---------------------------------- Office of General Counsel Date: 4/7/98 ---------------------------------- -8- EXHIBIT A [See Exhibit 10.10] EXHIBIT B (See attached) COLLEGE OF ENGINEERING THE UNIVERSITY OF TEXAS AT AUSTIN Department of Chemical Engineering, Austin, Texas 78712-1062 Adam Heller January 6, 1997 Ernest Cockrell, Sr. Chair Telephone: (512) 471-8874 FAX: (512) 471-8799 e-mail: heller@che.utexas.edu To: Dr. Paulette Braeutigam, Technology Licensing Coordinator From: Adam Heller Dear Paulette: This letter follows your discussion with Ephraim wherein you agreed to cover in the current agreement between The University of Texas at Austin and E. Heller & Company all presently existing intellectual property relating to biosensors that originated in my group. These include the following: US Patents Issued: ----------------- The already issued patents are: US 5,543,326 - Biosensor including chemically modified enzymes. US 5,356,786 - Interferant eliminating biosensor US 5,320,725 - Electrode and Method for the detection of hydrogen peroxide. US 5,264,105 - Enzyme electrodes. US 5,264,104 - Enzyme electrodes. US 5,262,305 - Interferant eliminating biosensor. US 5,262,035 - Enzyme electrodes. U.S. Patents Currently Prosecuted: --------------------------------- (1) Thermostable Biosensors ----------------------- The pending patent application discloses biosensors (methods and compositions) transducing biochemical concentrations into electrical signals at temperatures exceeding 37 (degree) C. The claims to be allowed are now negotiated with the Patent Examiner. Part of the material, where we report operation of a thermostable biosensor based on electrical connection of reaction centers of soybean peroxidase, was published [A Thermostable Biosensor of Hydrogen Peroxide, Vreeke, M.S., Yong, K.T., and Heller, A., Anal.Chem 67, 4247-4249 (1995)]. The hydrogen peroxide sensing biosensor operated continuously for weeks with no loss at 45 (degree) C and for hours at 75 (degree) C. The peroxide sensing layer of this biosensor was combined with enzymes that generated hydrogen peroxide in glucose and lactate sensors. The sensor of glucose operated for 10 days with only about 10% loss in activity and of lactate for about a week. A paper submitted is likely to appear in Analytical Chemistry in March, 1997. (Electrochemical Glucose and Lactate Sensors Based on "Wired" Thermostable Soybean Peroxidase Operating Continuously and Stably at 37 (degree) C, Kenausis, G., Chen., Q., and Heller, A. Submitted to Analytical Chemistry, 1996]. In the most stable group of sensors to date, we have immobilized the enzyme that catalyzes the reaction of oxygen with the analyte in a matrix containing silica, which we prefer to form by a sol-gel process from a molecular precursor of silica, such as a polyalkoxysilane. The immobilization of enzymes in sol-gel silica has been known from the work of others. We have, however, made two discoveries. First, that glycoprotein enzymes, such as glucose oxidase, can be made particularly thermostable by sol-gel immobilization in silica. Glucose oxidase, an example of a glycoprotein enzyme, withstands heating to 98 (degree) C for 10 min when immobilized. We have also discovered that while non- glycoprotein enzymes are much less stable in these matrices, they can be drastically stabilized in the sol-gel matrix by incorporating also a polymer. An example of such stabilization is that of lactate oxidase by poly(N-vinyl imidazole). When the two are co-incorporated in sol-gel silica, the lactate oxidase remains active after heating to 90 (degree) C for 10 min. We now know that different enzymes require different polymers for their stabilization in silica. The sol-gel silicas loaded with enzymes, that are hard solids, have been made into membranes and tested in glucose and lactate sensors. In these the oxidase enzymes generated the hydrogen peroxide, while electrically "wired" thermostable peroxidase transduced the hydrogen peroxide flux into a current by catalyzing the electroreduction of hydrogen peroxide to water. These exceptionally stable sensors have now also been miniaturized. They were incorporated in (galvanostatically etched) recesses in 0.3mm O.D. polymer insulated gold and palladium wires to form glucose sensors that, with mass transport controlling (e.g. dimethyl siloxane elastomer) layers that are permeable to oxygen and with a biocompatible layer of crosslinked (derivatized) poly(ethylene oxide) constitute our most stable in vitro and in vivo glucose sensors to date. From the in vitro data we project already that these systems will yield subcutaneously worn glucose sensors with useful lives exceeding one month. The above material will be subject to a continuation-in-part filing. Amperometric DNA and Oligonucleotide Sensors: --------------------------------------------- We have published a first paper on the sensing of hybridization of a model 30- base oligonucleotide as an electrical current [Direct Enzyme-Amplified Electrical Recognition of a 30-Base Model Oligonucleoutide, de Lumley-Woodyear, T., Campbell, C.N., Heller, A., J.Am.Chem.Soc. 118, 5504-5505 (1996)]. More recently we succeeded also in sensing of a natural 800-base oligonucleotide. We are currently considering whether to invest in a separate filing on the amperometric detection of oligonucleotide and DNA hybridization. A different application of the thermostable hydrogen peroxide sensors is in their use in selective sensing of oligonucleotides and DNA. For the first time we have in hand an electrically connected thermostable enzyme that can be used as a label of DNA strand. When its hybrid denatures or melts, the electrical connection of the thermostable peroxidase to the electrode surface is lost. Thus, the denaturing of an oligonucleotide or DNA hybrid (double helix) might be tracked with the thermostable enzyme. This would signal, through decline of the electrical current upon heating, the denaturing of the double strand and reveal the temperatures where denaturing starts and where it is complete. This temperature is known to be sensitive to the number of defects, i.e. the number of mismatched nucleotide pairs. In vivo Sensors: --------------- A patent application, some claims of which have been allowed, has been filed; continuations-in-part are planned. Innovations include: Components, physical structure and dimensions. The sensing layer is shielded and the sensors have at least two layer. One is a transducing layer, converting a biochemical flux into an electrical signal, that functions also in the absence of oxygen and has no leachable components. The second is a biocompatible layer that swells in an aqueous solution, increasing its volume by at least 20%. The preferred embodiment has three layers, the central layer limiting the mass transport of the analyte and thereby expanding the range of concentrations measured. The preferred sensors have diameters smaller than 0.5 mm, preferable smaller than 0.3 mm and are subcutaneously implanted. Papers published including part of the material on these sensors are: Design and optimization of a selective subcutaneously implantable glucose electrode based on "wired" glucose oxidase, Csoregi, E., Schmidtke, D.W. and Heller, A. Analytical Chemistry, 67, 1240-1244 (1995). Design, Characterization, and One- Point In Vivo Calibration of a Subcutaneously Implanted Glucose Electrode, Csoregi, E., Quinn, C.P., Schmidtke, D.W., Lindquist, S.-E., Pishko, M.V., Ye, L., Katakis, I., Hubbell, J.A. and Heller, A., Analytical Chemistry, 66, 3131- 3138 (1994). In vivo sensors of this type, containing a thermostable transduction layer were discussed above. -2- Another type of novel sensor was made by placing a non-corroding wire, coated in part with a transducing layer, and having no leachable component in a microdialysis or microfiltration fiber, made of a biocompatible material. Preferably, the transducing layer is overcoated with a mass transport limiting membrane. The overall diameter is about 0.5 mm or less. Polysulfone microfiltration fibers and cellulose acetate/polysulfone mass transport limiting layers were used. The sensing systems may have a skin electrode, i.e. a counter-reference electrode, such as a silver/silver chloride electrode, on the skin. The use of skin electrodes was described in our papers, Statistics for Critical Clinical Decision Making Based on Readings of Pairs of Implanted Sensors, Schmidtke, D., Pishko, M.V., Quinn, C.P., and Heller, A., Analytical Chemistry, 68, 2845-2849 (1996), and also in Kinetics of Glucose Delivery to Subcutaneous Tissue in Rats Measured with 0.3mm Amperometric Microsensors, Quinn, C.P., Pishko, M.V., Schmidtke, D.W., Ishikawa, M., Wagner, J.G., Raskin, P., Hubell, J.A. and Heller, A., Am.J.Physiol. 269 (Endocrinol.Metab. 32) E155-E161 (1995). On the basis of the information contained in our paper Statistics for Critical Clinical Decision Making Based on Readings of Pairs of Implanted Sensors, Schmidtke, D., Pishko, M.V., Quinn, C.P., and Heller, A., Analytical Chemistry, 68, 2485-2489 (1996), we are seeking patent protection on using in vivo redundant sensors; on the method of selecting pairs of sensor readings that are valid for one-point calibration in vivo, discriminating these from those that are not; and for defining pairs of sensor readings that will trigger an alarm signaling hypoglycemia, hyperglycemia, excessively fast rise, or excessively fast decline of glucose levels, or of sensor malfunction and statistical algorithms for the selection of valid readings. We are also seeking to patent the method of calibrating a sensor in vivo by withdrawal of one sample of blood and its independent analysis described in the papers of Csoregi, E., Schmidtke, D.W., and Heller, A. Analytical Chemistry, 67, 1240-1244 (1995); and of Csoregi, E., Quinn, C.P., Schmidtke, D.W., Lindquist, S.-E., Pishko, M.V., Ye, L., Katakis, I., Hubbell, J.A. and Heller, A., Analytical Chemistry, 66, 3131-3138 (1994). We have shown that after perturbing the venous glucose concentration by injection of glucose or insulin, the change in the subcutaneous glucose concentration lags behind the blood concentration. For glucose injection the lag is glucose dose dependent (Kinetics of Glucose Delivery to Subcutaneous Tissue in Rats Measured with 0.3mm Amperometric Microsensors, Quinn, C.P., Pishko, M.V., Schmidtke, D.W., Ishikawa, M., Wagner, J.G., Raskin, P., Hubbell, J.A. and Heller, A., Am.J.Physiol. 269 (Endocinol.Metab. 32) E155-E161 (1995)) and is in the 9-13 min range; for insulin injection the lag is much longer (15-40 min) (David W. Schmidtke, Ph.D. Thesis, The University of Texas at Austin, December, 1996). We shall seek to protect the algorithms relating blood with subcutaneous glucose concentrations. We have observed that after insulin injection the dynamic difference between subcutaneous and blood glucose concentration is high. Our method will exclude a period of about 30-90 min., preferably of about 40 min. following an insulin injection from the periods in which the sensor is calibrated in vivo. We have published work on miniature electronics, including a biopotentiostat, for glucose sensing in vivo [Battery Powered Miniature Bioporentiostats for Amperometric Biosensors, Quinn, C.P., Wagner, J.G., Heller, A., and Yarnitzky, C.M., Instrumentation Science & Technology, 24, 263-275 -3- (1996)] and are currently building a system with a miniature transmitter. The electronics have been miniaturized to be wrist-worn. We have shown that there are preferred sites for implanting glucose sensors in humans. One of the preferred sites is the back of the hand above the knuckles and below the wrist. The sensors' output when in the hand or another extremity is (outside) temperature dependent, wherefore an algorithm correcting for the sensor temperature is useful. We have recently demonstrated that a useful biocompatible layer for our in vivo sensors can be made by crosslinking a polyamine, such as an amineterminated dendrimer, wherein the crosslinkers may be polyethylene oxide chains, the polyethylene oxide having amine-reactive and functions such as active esters of N-hydroxy succinimide. This adds to our claims of biosensors that are made with a biocompatible gel that swells in water, to include gels made by reacting two materials, at least one of which has poly(ethylene oxide) chains. Sensors for Bioreactors: ----------------------- We have built and tested sensors for bioreactors. The glucose sensor built does not require oxygen for its operation. It is placed in a sterilizable sleeve having a non-fouling membrane. The sensor was designed to minimize the volume of the fluid contained in the sleeve's compartment behind the sterilizable membrane in which the periodically replaced sensor is sited. Sensors were replaced without risking infection of the broth with a foreign organism. Air Oxidizable Redox Polymers and Redox Couples: ------------------------------------------------ We have observed that on some redox polymers used to connect enzymes to electrodes molecular oxygen can be catalytically electroreduced. The importance of such electroreduction is in that it provides another method for sensing oxygen amperometrically, but, more importantly, in allowing maintenance of the redox centers of the polymer predominantly in their oxidized state simply by storing the biosensors in air or in any other oxygen containing atmosphere. We have found that electrodes modified with poly(N-vinyl imidazole), and preferably with the [Os(bpy)2 CL] +/2+ complex (where bpy is bipydrine, phenantholine or a derivative of these) of poly(N-vinyl imidazole) are oxygen reduction catalysts. As a result of the Os redox centers in these are maintained dominantly as in the Os(III) oxidation state. In general, the more reducing the Os complexes bound to poly(N-vinyl imidazole), the better they catalyze O2 reduction. Thus (Os(dmo'bpy)2 Cl] +/2+ complexed poly(N-vinyl imidazole), where dmo-bpy is 4,4'- dimethoxy-2,2'-bipyridine is an excellent O2 reduction catalyst. Less effective catalysis of O2 reduction was observed also with poly(4-vinyl pyridine) complexes of osmium and with osmium ligands like 4,4'-dimethyl-2,2'-bipyridine. It is possible that non-polymeric imidazole complexes, such as [Os(Im)(dmo-bpy)2 Cl] are also maintained by oxygen in the oxidized state, [Os(Im)(dmo-bpy) 2 Cl] +. The ability to maintain Os centers in the oxidized state is an advantage in biosensors where charge passed is measured or where the current after a defined elapsed time is measured. The signal from such sensors is less dependent on storage (residual enzyme activity) or temperature. -4- Redox Polymers and Synthesis of Polymers: ---------------------------------------- New enzyme wiring redox polymers were described in four articles [(a) 'Wiring' of glucose oxidase and lactate oxidase within a hydrogel made with poly(vinyl pyridine) complexed with [Os(4,4'dimethoxy-2,2'-bipyridine)2 Cl]+/2+ Kenausis, G., Taylor, C. and Heller, A., J. Chem. Soc., Faraday Transactions, 92, 4131- 4136 (1996); (b) "Wiring" of Glucose Oxidase within a Hydrogel made with Polyvinyl Imidazole Complexed with [(Os-4,4'-dimethoxy 2,2'-bypyridine)Cl]+/2+ Chris Taylor, Gregg Kenausis, Ioanis Katakis, and Adam Heller, J.Electroanalytical Chemistry 396, 511-515 (1995); (c) Polyacrylamide Based Redox Polymer for Connecting Redox Centers of Enzymes to Electrodes, De Lumley- Woodyear, T., Rocca, P., Lindsay, J., Dror, Y., Freeman, A., and Heller, A., Analytical Chemistry 67, 1332-1338 (1995); (d) Glucose Electrodes Based on Crosslinked [OS(bpy)2Cl]+/2+ Complexed Poly(1-Vinylimidazole) Films, Ohara, T.J., Heller, A., Anal.Chem. 65, 3512-3517 (1993)]. Some of the new polymers were designed to have redox potentials shifted in the negative direction, so as to reduce the unwanted electrooxidation of easy to oxidize constituents of biological fluids, like ascorbate and acetaminophen. Others were designed to reduce the positive charge on the chains, so as to lessen the permeation of interfering anions (ascorbate, urate) and the non-specific binding of biologically important polyanions (oligonucleotides, DNA, some enzymes). It was found that the quality of the electrical connection of redox enzymes to electrodes critically depends on the extent of branching and crosslinking of the enzyme-wiring redox polymers. Such branching and crosslinking takes place when in polymer-nitrogen bound [Os(bpy)2 Cl]2+/+ centers, denoted as poly-[N - OS(bpy)2 Cl]+/2+ the chloride in the inner coordination sphere is exchanged by a second nitrogen of polymer chain. Branching and crosslinking occur by formation of poly [N - Os(bpy)2- N]2+/1+ embranes poly links. By introducing rigorously anhydrous synthetic and storage conditions and by monitoring the progress of the desired complex formation in its preparation, dominantly linear polymers that "wire" the enzymes more effectively were prepared. New Crosslinkers: ---------------- Novel crosslinkers have been used. These have usually a poly(ethylene oxide) sequence, terminated by functions reacting with amines such as N- hydroxysuccinimide esters, suberimidates, dichlorotriazine etc. Others are similar, but have polymethylene chains. Through using these crosslinkers we are making hydrogels that swell less, adhere better to electrodes, and are less subject to loss from the electrode surface by shear. Novel Enzymes and their "wires": ------------------------------- In collaboration with colleagues at the University of Glasgow, Scotland, we have started work on glutamine electrodes based on wiring of glutamine oxidase. At Austin and also in collaboration with colleagues at the University of Lund in Sweden on wiring oligosaccharide dehydrogenase we used polymeric "wires" that do not have a net positive charge and on polymers having anionic and cationic zones. Membranes: --------- -5- The formation of membranes to (a) extend the sensing range of electrodes; (b) reduce the electrooxidation of interferants; (c) control the ratio of the fluxes of O2 and the analyte (e.g. glucose) to the electrode surface has been studied. Methods of casting miniature membranes (e.g. of cellulose acetate from cyclohexanone, of polysulfone from tetrahydrofuran) and of their mixtures, and also of crosslinking polydimethyl siloxanes to form oxygen "breathing" elastomeric micromembranes were developed. Immunosensors: ------------- We have built immunosensors based on wiring of immunolabeling enzymes. In one group of these, no separation or washings are required; in others the substrate of the immunolabeling peroxidase, hydrogen peroxide, is generated in situ by air oxidation of a substrate such as choline with an immobilized enzyme such as choline oxidase. Choline oxidase, as well as a small number of other oxidases is not "wired" in our hydrogels, thus performing its usual catalysis without interfering with the amperometric assay. An example of the immunosensors developed is an electrode modified with a film consisting of a redox hydrogel capable of "wiring" a peroxidase, an antibody and choline oxidase. The electrode is exposed to the solution tested for antigen, then to a peroxidase labeled antibody solution also containing choline. When the antigen is present, "sandwich" formation causes electrical connection of the labeling peroxidase and electroreduction of the H2 O2 generated in the air oxidation of choline to betaine. Antibodies are detected through incorporation of antigens in the redox polymer and choline oxidase containing gel, exposure to the solution tested for the antibody, to which choline and the peroxidase-labeled antigen were added. With my regards and best wishes for 1997, /s/ Adam Heller Adam Heller AH:n -6-