Hydrogel biosensor

Details Hydrogel biosensor Hydrogel biosensor

2001-04-20

2003-02-04

Redding, David A. (Department: 1744)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S287100, C435S287700, C604S891100, C604S892100

Reexamination Certificate

active

06514689

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to biosensors utilizing hydrogels to measure the concentration of free analyte molecules in a fluid, particularly biosensors suitable for implantation in a patient to provide constant monitoring of a selected analyte. The invention also relates to health alarm systems in which a biosensor is connected to apparatus which alerts a patient and/or patient caretakers to deleterious changes in the levels of analyte in the patient's body fluids or to other adverse changes in a patient's condition.
2. Description of Related Art
During the past decade, intense effort has been directed toward the development of analyte monitoring biosensors as an aid to prevent complications of diseases such as diabetes. Development of an implantable analyte sensor that is specific and sensitive enough to precisely and continuously monitor analyte levels in vivo would be a significant advance. Such a sensor would also greatly facilitate data collection and biochemical research relating to analyte levels in patients.
Several new implantable techniques have been developed for analyte analysis such as glucose in clinical practice based on electrochemical principles and employing enzymes such as glucose oxidase (GOD) for analyte recognition. Potentially implantable analyte biosensors based on electrochemical transducers are the most highly developed, and this class of sensors can be further subdivided into potentiometric sensors, conductometric sensors, and amperometric sensors. At present, neither the potentiometric method nor the conductometric method appears to be suitable for in vivo analyte monitoring due to: (a) interference by species other than analyte in the physiological environment; (b) low sensitivity and logarithmic dependence of the signal on the analyte concentration. A linear dependence of the signal on analyte concentration is highly desirable because of the need for repeated recalibrations over time for implanted analyte sensors. However, non-linear calibration curves can be handled reasonably well using microprocessors.
The most advanced analyte sensors for in vivo monitoring are electrochemical sensors containing hydrogels in which an enzyme which generates hydrogen peroxide upon reaction with the analyte is immobilized, with an amperometric method being used to detect the hydrogen peroxide. This technique offers the possibility for a linear calibration curve. In the amperometric method, an electrode is used which produces a current proportional to the diffusional flux of hydrogen peroxide (H
2
O
2
) to the electrode surface, or, alternatively, proportional to the diffusional flux of oxygen (O
2
) to the electrode surface. An increase in the surrounding analyte concentration should increase the diffusional flux of analyte into the membrane and increase the reaction rate within the membrane. The increase in enzymatic reaction rate in turn should increase the local hydrogen peroxide concentration and decrease the local oxygen concentration within the membrane. This should lead to an increase in the current detected by a hydrogen peroxide-based electrode sensor, or a decrease in current as detected by an oxygen-based electrode sensor. The latter approach, based on detecting the oxygen flux, also requires a second oxygen-based electrode sensor located in a hydrogel without the enzyme. This second electrode is used as a reference.
However, amperometric sensors must overcome several hurdles before they will ever be useful for commercial in vivo monitoring. Current analyte sensor designs appear unlikely to solve these difficult problems in the near future. The first hurdle arises from electrochemical interference. The analyte (whether hydrogen peroxide or oxygen) must be the only species present which produces a current at the electrode. Hence for both oxygen-based and hydrogen peroxide-based analyte sensors, an inner membrane must be used which is permeable to the analyte but impermeable to endogenous interferents which may produce electrochemical effects. In clinical studies of the hydrogen peroxide-based sensor, a decay in sensitivity over the implant period was observed, a phenomenon which could not be explained by blockage of the sensor surface by protein. One possible explanation for the loss of sensitivity is hydrogen peroxide mediated enzyme deactivation. For the oxygen-based sensor, this can be avoided by co-immobilizing catalase with enzyme, because catalase consumes hydrogen peroxide. Fourthly, a shortage of oxygen relative to analyte can place an upper limit on the biosensor's ability to measure analyte levels. This problem is called the “oxygen deficit”.
In addition to the biosensors described above, hydrogels have also been used in devices developed to release insulin directly into a diabetic's bloodstream in response to high analyte levels. In one approach, the hydrogel is constructed to have chemically immobilized pendant groups which are charged under physiological conditions (pH2 to pH10). Molecules of glucose and of a glucose-specific binding molecule (abbr. GBM; for example, concanavalin A) are also immobilized in the gel. Within the hydrogel, in a solution which contains no free glucose, immobilized glucose molecules bind to immobilized GBMs, forming what are in effect non-covalent ‘crosslinks’. As the hydrogel is exposed to a fluid containing free glucose, binding competition displaces immobilized glucose from GBMs, thus reducing the number of ‘crosslinks’. This reduction in crosslinking causes an increase in swelling of the hydrogel, due to the presence of the charged pendant moieties in the hydrogel. In effect, the hydrogel swelling increases the porosity and/or pore size between gel subunits. These insulin-delivery hydrogels also contain insulin, and the increase in pore size in turn allows insulin (a rather large molecule which does not diffuse readily through a closely-crosslinked gel matrix) to diffuse outward and be released into the patient's bloodstream. See A. Obaidat, et al., Characterization of Protein Release through Glucose-sensitive Hydrogel Membranes, 18 BIOMATERIALS 801-806 (1997); Y. Ito, et al., An Insulin-releasing System that is Responsive to Glucose, 10 JOURNAL OF CONTROLLED RELEASE 195-203 (1989), which are expressly incorporated herein.
However, so far as we are aware, the changes in swelling force/osmotic pressure that occur in pH-sensitive competition binding hydrogels have not heretofore been recognized and exploited for the measurement of the concentration of free analyte. The prior art does not teach measurement of the analyte-induced swelling of the hydrogel as a method of measuring analyte concentrations, and it specifically does not teach the use of a transducer to measure hydrogel swelling. The use of a pressure transducer provides a measurement tool that avoids the problems encountered by electrochemical sensors.
Thus, a need exists for a biosensor that is extremely sensitive to the concentration of analyte, and also relatively free from interference, even when operating in complex media such as human blood. A need further exists for a biosensor that relies directly on change in analyte, since analyte concentration itself is a much more controlled parameter than parameters measured by electrodes. This is especially critical in implantable biosensors, because this system is relatively free from potential sources of interference. Additionally, there is a need for hydrogel-based biosensors in which the analyte-detecting process does not consume oxygen.
Accordingly, it is the object of this invention to provide such a biosensor. It is a further object to provide a general method for measuring the concentration of any analyte for which a suitable specific binding partner can be found or constructed.
SUMMARY OF THE INVENTION
The present invention comprises a hydrogel-based biosensor which measures the osmotic pressure within a hydrogel having pendant charged moieties, analyte molecules, and analyte binding partner molecules all immobi

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