WO2008045145A2

WO2008045145A2 - Novel bio-composites for sensors and processes for producing the same

Info

Publication number: WO2008045145A2
Application number: PCT/US2007/013080
Authority: WO
Inventors: Pelagia-Irene Gouma
Original assignee: The Research Foundation Of State University Of New York
Priority date: 2006-06-06
Filing date: 2007-06-01
Publication date: 2008-04-17
Also published as: WO2008045145A3

Abstract

A process for producing bio-composite oxide thin films intended for the use as urea biosensors is provided in this application. The encapsulation of bio-sensitive molecules/cells, such as bacteria, cells, enzymes, in the inorganic matrix of the sol-gel processed molybdenum trioxide and the retention of the bio-sensitive molecule/cell is described herein. This is important to develop resistive bio-detectors where the gas sensitive matrix measures the products of the biochemical reaction (i.e., gaseous ammonia) of the hydrolysis of urea by urease. The invention is also directed to the bio-composite oxide thin films produced by the described process.

Description

NOVEL BIO-COMPOSITES FOR SENSORS AND PROCESSES FOR PRODUCING THE SAME

PRIORITY

This application claims priority to application serial number 60/811,162, which was filed

with the U.S. Patent and Trademark Office on June 6, 2006.

GOVERNMENT SUPPORT

The invention was supported, in part, by a grant DMR0224642 from the National Science Foundation. The U.S. Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to a sensing platform for medical diagnostics

based on the incorporation of biomolecules (receptors) in active, gas sensitive matrices.

BACKGROUND OF THE INVENTION

Developing sensors that are dependable and sensitive enough to be used in the medical

and/or agricultural industries, among other industries, is of considerable interest to these fields.

Potentiometric biosensors that have been used for these purposes have been used in the past, but are slow to respond to the presence of a particular chemical species as well as to respond to the

change in concentration of the species. In addition, these types of sensors take even more time to

recover than it takes to respond.

Described herein is a sensing platform for medical diagnostics based on the incorporation

of biomolecules (receptors) in active, gas sensitive matrices. The receptor biomolecules may be

enzymes, bacteria, cells that induce biochemical reactions with the analytes of interest, resulting

in the release of gases. The gaseous species are detected rapidly by the active matrix

compounds, typically chemoselective metal oxides or polymers, hi this work, the development of dio-doped transition metal oxide-based detectors for monitoring metabolic products (such as urea) in aqueous media is discussed. The advantage of the proposed sensor technology over existing biosensors lies in the rapid and selective detection of bio-species and other organic as well as inorganic molecules by solid state devices using electrical signals. The matrices of choice are selective gas sensing probes utilizing particular polymorphs of the MoO₃ and WO₃ systems that respond to NH₃ and NO_x, respectively.

Several different types of sensing platforms are described herein, each of which overcome the shortcomings of existing sensing platforms available on the market today, hi other words, the sensing platforms described herein provide rapid detection of pathogens and other bio-species with inherent specificity.

SUMMARY OF THE INVENTION

The present invention generally relates to a sensing platform for medical diagnostics based on the incorporation of biomolecules (receptors) in active, gas sensitive matrices. More specifically, the invention relates to the use of the arrays of biocomposite and bio-doped films to detect the chemical products of biochemical reactions, such as ammonia, NO, is described herein.

DETAILED DESCRIPTION OF THE FIGURES

The above and other objects, features and advantages of certain exemplary embodiments of the present invention will be more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

Figure 1 is a graphical representation of the response of an olfactory system to NO of the present invention; Figure 2 is a graphical representation of the activity of urease encapsulated in MoO₃ sol- gel of the present invention;

Figure 3 is a design of an electronic tongue element using the encapsulated sensors of the present invention;

Figure 4 shows a TEM micograph of the hybrid urease - MoO₃ gel;

Figure 5 is a graphical depiction of the change in potential upon reaction of urea with enzyme-metal oxide gel;

Figure 6 shows the morphology (SEM) of the sol-gel film with urease encapsulated within it;

Figure 7 shows the TEM image taken at higher magnification (125KX) showing clusters of molybdenum oxide formed upon drying of the sol-gel;

Figure 8 is a graphical representation of the concentration of urea versus activity plot;

Figure 9 is a graphical representation of the activity of dried sol-gel (xerogel) versus the concentration of urea;

Figure 10 is a SEM of polymer-enzyme nanofibers;

Figure 11 is a SEM of nanowoven electrospun composite mats of urease; and

Figure 12 is a graphical representation of polymer-enzyme composition reacting differentially concentrated urea solutions.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A description of detailed construction of preferred embodiments is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope and spirit of the invention. Descriptions of well-known functions and constructions are omitted for clarity and conciseness.

The use of enzymes, cells, bacteria, DNA, RNA, proteins, antibodies etc. are used in polymer or inorganic matrices to detect, measure and/or monitor chemical species or gaseous byproducts produced by the species are described herein. For example, yeast cells were entrapped in SiO₂ sol, trypsin in SiO₂/TMOS. Development of biosensors is the most widely researched application for the hybrids discussed. Glucose oxidase was immobilized in SO₂/TEOS for electrochemical detection of O₂ where raising the aging temperature increased the yield, but lowered the activity. Urease was encapsulated in TiO₂-Cellulose for a bio-sensing application with a long response time>30 minutes. Urease in TEOS was studied as a UV-VIS biosensor with urea detection limit of 0.5mM. The majority of the sensors utilize bio-recognition elements as physical transducers to convert biological reaction into a measurable signal. These sensors can be used in the fields of agriculture, food chemistry, medicine, defense, and/or medicine. Other uses not mentioned herein are also within the scope of the invention.

Enzymes are nature's most specific and selective catalysts and many of them have been identified as precise bio recognition molecules applicable in the sensing field. The greatest obstacle preventing a large sealed production of enzyme-based sensors is the loss of enzyme activity in even slightly non-biocompatible environments. Innovative urea biosensors obtained through electrospinning nano fibers of urease and polymer composite, and employing the sol gel method to encapsulate the enzyme inside metal oxide semiconductor thin films are described herein. The large amount of available surface area obtained through both methods has the potential to provide unusually high sensitivity and fast response time in sensing applications. A one enzyme in particular, urease E.C.3.5.I.5., acts as a catalyst in the hydrolysis or urea to ammonia and carbon doxide. Urea is one of the main components of human urine, and a waste product that builds up in the human blood. Abnormal levels of urea can indicate liver function problems. Therefore, it has found a wide range of applications in the medical field for detoxifying blood in kidney machines.

In one embodiment of the invention, as enzyme-polymer solution was prepared by mixing 70% by volume of 4.615* 10^'5M polyvinylpyrrolidone (PVP) in ethanol solution, with 30% by volume urease solution with 1577.6 units of urease dissolved in 1OmL of .1M PBS buffer. Reactivity measurements were taken for five differently concentrated urea solutions using the Thermo Orion ammonia electrode with the urease/polymer solution before and after electrospinning. The increase in ammonia concentration for both the solution and electrospun fiber mats proved that the enzyme retained activity not only inside the polymer solution, but also though the electrospinning process. For the sol gel encapsulation a .1M MoO₃ sol gel was divided into two parts. In both parts 2ml of urease solution was added (1577.6 units in water and glycerol). Where one part added it before ultrasonication and the other after one hour of ultrasonication, both of them were in mixed for a total of two hours. Both mixtures retained enzyme activity, and acted as catalysts in the hydrolysis.

In another embodiment of the invention, gas sensitive matrices were developed using sol- gel technology, including WO₃, MoO₃ and a hybrid of TiO_2-MoO₃. A modified electronic alfactory system (based on the design of SACMI EOS835) was used for testing the gas selectivity of these sensor matrices. Figure 1, for example, shows the individual sensor response of the three-sensor array to NO. Bio-doped oxides were prepared by adding enzymes to the sol-gel matrices. Figure 2 gives an example of activity measurements carried out in MoO₃ encapsulated urease verifying that the bio-molecules preserve their activity while entrapped in the organic matrices.

Also provided here (Figure 3) is the initial design of an electronic tongue element for a system utilizing a urea sensing probe; other probes include bacteria sensors.

In still another embodiment of the invention, synthesis of bio-doped oxides for urea sensing have been produced using the procedures described herein below. As stated above, developing a urea sensor is of considerable interest owing to its demand in medical and agricultural industries, as amount of urea in blood is indicator of kidney function, whereas 2.8- 7.1 mmol/L of urea concentration in blood is normal, 50 mmol/L of urea is critical value. Among the different types of urea sensors currently available, e.g., amperometric, conductimetric, FET-based, and potentiometric, the latter has emerged as the most widely used detector due to the general availability of the instrumentation. This involves urease immobilized in passive media, such as natural and synthetic polymers, and a probe electrode (e.g., pH electrode). Potentiometric bio-sensors, however, are slow to respond to the presence of urea and to changes in its concentration and even slower to recover. Total detection time can be up to several minutes. Furthermore, interference OfNa⁺ and K⁺ ions to the NH₄ ion electrode is another drawback.

As mentioned above, therefore, there is still a need for a urea sensor which is selective, fast, robust and reproducible. One approach to make such a sensor is based on encapsulating the enzyme (urease) that specifically catalyses the hydrolysis of urea releasing gaseous ammonia in a gas sensitive (active) matrix. Sol-gel processed molybdenum trioxide (MoO₃) was chosen as the porous matrix to host urease. Orthorhombic MoO₃ was found in our earlier studies to be a highly specific ammonia sensing element; its electrical resistance changing appreciably in the presence of even traces of ammonia gas with a response and recovery time of a few seconds. Although enzyme encapsulation in silica (transparent glass) matrices has been explored by several groups, this is the first time that a non-transparent and active oxide matrix is used to incorporate biomolecules for biosensing applications.

The urease bio-doped oxides for urea sensing discussed above can be made using the following procedures. Urease solution was made by mixing 0.662g (1059 units) of urease ((EC 3.5.1.5) from SIGMA) in 25ml water + 25 ml glycerol. This procedure was based on the procedure of JHA, P, et al., "Nanostructured materials for sensors" Nano-2004 India: international conference on nano-materials, November 2004. The solution or encapsulating the urease in sol-gel, 0.39104g of molybdenum isopropoxide (purchased from CHEMAT TECHNOLOGY INC.) was added to solution containing 7 ml butanol (Butanol reagent ACS 99.4%(GC) from ACROS), 2 ml of 0.1 M PBS buffer (PBS saline P-3183 from SIGMA) and 1 ml of urease solution. The mixture was placed in the ultrasonic cleaner for 2 hours and the sol was allowed to settle for 2 days in the refrigerator. Transmission Electron Microscopy studies confirmed that enzyme clusters were entrapped within pores of the gel (see Figure 4). In order to assess the retention of the activity of urease in the sol-gel, standard urea test solution were prepared. Thus, 1 ml of sol was mixed with 20 ml of urea solution and 1 ml of ammonia pH adjusting ISA, then it was stirred by magnetic stirrer for 15 minutes. Five minutes later the voltage readings were taken using Thermo Orion ammonia electrode (see Figure 5).

To ensure that the activity detected was specifically due to the enzyme encapsulated and not because of the presence of other solution interfering chemicals, the individual effect of each component was considered. PBS caused the electrode potential to increase while pH adjusting ISA decreased the value of the electrode potential. Therefore, for all the readings the measured value of the potential after adding ISA was recorded. The value of the enzyme activity was calculated as the change in potential after adding sol-gel compared to the value of potential after adding ISA. Finally, the effect of film drying on the enzyme's activity was studied. Spin coated films of the composite sol-gel were dried at room temperature for 3 days. Samples were made by varying the amount of PBS from 2ml to up to 4ml.

Figure 6 shows the morphology of the sol-gel film with urease encapsulated within it. The image shows the surface structure of the sol-gel film containing biomolecules. Figure 7 shows an additional view of a TEM image taken at higher magnification (125kX) showing clusters of molybdenum oxide formed upon drying of the sol-gel. These aggregates are porous as observed from the image. The urease molecules were seen to be encapsultated inside the pores. The size of the individual particles in each cluster is around 10-20 nm.

Urease retained its reactivity (to catalyze the hydrolysis of urea) inside the molybdenum trioxide sol-gel. Figure 8 shows the concentration of urea versus activity plot which shows steady increase in activity with increase in urea concentration. The urea test solutions had concentrations in the range of ImM so as to be able to detect urea levels that are below the "critical value" of urea in blood.

The urease retained its reactivity even after the sol-gel was dried. Figure 9 shows the activity of dried sol-gel (xerogel) versus the concentration of urea. The observed reduction of the activity in the xerogels might be attributed to anyone of the following factors: (i) inefficient retention of enzyme by the porous gel; (ii) possible collapse of the pore openings as the sample during drying; (iii) partial denaturing of the enzyme during the drying process. Further studies are currently underway to optimize the condition of enzyme encapsulation and retention in these xerogels.

Measurements were also made by varying the amount of PBS. It has been observed that changing the amount of buffer did affect the activity, but did not cause any major reduction in it.

When bio-doped MoO₃ gels were prepared, it was observed that the enzyme retains its reactivity inside the sol-gel. The success of this step was vital for the synthesis of a resistive biosensor. The presence of the matrix consisting of the molybdenum oxide phase around the urease moleculdes is expected to greatly reduce the diffusion time of ammonia liberated from the hydrolysis of urea, to the metal oxide. The ammonia liberated can be detected in-situ, thus giving very fast response times.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A process for preparing bio-composite oxide thin films comprising the steps of: (i) encapsulating an enzyme in sol-gel by adding molybdenum isopropoxide to a solution comprising butanol, PBS buffer and an enzyme solution;

(ii) subjecting the solution produced in step (i) to an ultrasonic motion for a time effective to produce an ultrasonic treated solution;

(iii) refrigerating said ultrasonic treated solution of step (ii) for a predetermined amount of time to produce a sol-gel comprising an encapsulated enzyme, bio- composite oxide thin film.

2. The process of claim 1, wherein the enzyme is urease.

3. A bio-composite sensor thin film comprising a biomolecular receptor entrapped within the pores of the matrix of said thin film, wherein said biomolecular receptor is an enzyme, and said matrix comprises a metal oxide.

4. The bio-composite sensor of claim 3 wherein said enzyme is urea and said metal oxide is sol-gel processed molybdenum trioxide.

5. Bio-doped molybdenum trioxide sol-gels, wherein said sol-gels comprise a multitude of pores, and wherein said pores contain biomolecular receptors entrapped therein.

6. The bio-doped sol-gels of claim 5 wherein said biomolecular receptors are selected from enzymes, bacteria, DNA, RNA, proteins, antibodies, cells that induce biochemical reactions with the analyte of interest resulting in the release of gases, and mixtures thereof.

7. The bio-doped sol-gels of claim 6 wherein said biomolecular receptor is an enzyme.

8. The bio-doped sol-gels of claim 7 wherein said enzyme is urease.

9. A medical diagnostic device comprising the bio-composite sensor of claim 3.

10. A bio-composite thin film produced in accordance with the process of claim 1.

11. A process for preparing bio-composite oxide thin films comprising a biomolecular receptor entrapped within the pores of the matrix of said thin film, said process comprising the steps of:

(iv) encapsulating a biomolecular receptor within a sol-gel by adding a polymorph selected from molybdenum isopropoxide or WO₃ to a solution comprising butanol, PBS buffer and a solution of said biomolecular receptor;

(v) subjecting the solution produced in step (i) to an ultrasonic motion for a time effective to produce an ultrasonic treated solution;

(vi) refrigerating said ultrasonic treated solution of step (ii) for a predetermined amount of time to produce a sol-gel comprising an encapsulated, bio-composite oxide thin film having biomolecular receptor entrapped within the pores of the matrix of said thin film.

12. The process of claim 11 wherein said biomolecular receptors are selected from enzymes, bacteria, DNA, RNA, proteins, antibodies, cells that induce biochemical reactions with the analyte of interest resulting in the release of gases, and mixtures thereof.

13. The process of claim 12 wherein said biomolecular receptor is an enzyme.

14. The process of claim 13 wherein said enzyme is urease.

15. The process of claim 11 wherein said polymorph is molybdenum isopropoxide.