[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
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(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.
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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:
---------
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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
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