PLASTIC BIOCHIP BASED ELECTROCHEMICAL IMMUNOSENSOR

Field of the Invention

The invention relates generally to electrochemical biosensors and particularly to biosensors for detecting disease markers or environmental pollutants in body fluids.
This complete specification takes priority from provisional patent application No. 236/CHE/2010 filed on February 1, 2010.

Description of the Related Art

Immunoassays are commonly performed today for the screening and detection of various disease markers or environmental pollutants. Immunoassay detection is typically performed by optical, electrochemical and impedimetric ways. Optical and impedimetric detection require complex instrumentation and are thus limited for use in laboratories, while electrochemical detection of immunoassays is simple and low cost and is gaining popularity for use in point-of-care applications along with the development of hand-held devices. One of the technological limitations for commercializing electrochemical detection for biosensors and immunosensors is lack of a single use, low cost and highly sensitive electrochemical sensor. Current point-of-care equipment manufacturers such as l-STAT and Affymetrix use a silicon gold chip as an electrochemical sensor which requires highly sophisticated manufacturing environment and the silicon chip is very expensive even at very large quantities of one million pieces. Some other point-of-care manufacturers use screen printed electrochemical sensors comprising of carbon electrodes. Carbon electrodes, while being low cost, do not have high sensitivity and their use is limited to applications where sensitivity requirements are not high.

Existing prior art immunosensor devices exemplified by US patent application no. 20060134713 and US7455756 are complicated multiple layer devices that entail relatively high manufacturing cost, while glucose sensor devices such as disclosed in US7829023, US6287451 and US20030155237 are disposable strip devices that have multiple layer construction and although disposable, are also of complex design.
Thus there exists a need for a single use disposable, highly sensitive and low cost electrochemical sensor for biosensor and immunosensor applications.

Summary of the Invention

A disposable electrochemical immunosensor is disclosed, comprising a polyester substrate with a sputtered metallic layer on one surface thereof, a sensor comprising a working electrode, a reference electrode and a counter electrode formed on the metallic layer. A polymer film spacer comprising an opening is adhesively bonded onto a portion of the metallic layer such that the opening overlays the working electrode, reference electrode and counter electrode of the sensor. The sensor electrodes on the metallic layer and the opening on the polymer film spacer are formed by laser ablation. A biological protein layer comprising antibodies modified to include thiol groups is immobilized within the cell formed by the opening on the polymer film spacer over and in contact with the electrodes.

In alternative embodiments, the metallic layer of the electrochemical immunosensor can be of gold or platinum or palladium.

Optionally, the reference electrode of the electrochemical immunosensor can be electroplated with silver-silver chloride.

The electrochemical immunosensor can be used to perform both sandwich-type and competitive type immunoassays by immobilizing antibodies for the target molecule within the electrochemical cell.

In one embodiment, the electrochemical immunosensor is treated with secondary antibodies labeled with alkaline phosphatase, horseradish peroxidase (HRP) or glucose oxidase.

In one embodiment, the electrochemical immunosensor is configured for detection using a substrate reagent comprising glucose and either potassium ferricyanide or hexamine ruthenium chloride as mediator.

In one embodiment, the sensitivity of the immunoassay by the electrochemical immunosensor is enhanced by use of carbon nanotubes deposited on the working electrode.

Brief Description of the Drawings

The invention has other advantages and features which will be more readily
apparent from the following detailed description of the invention and the
appended claims, when taken in conjunction with the accompanying drawings, in
which:

Figure 1A shows the laser ablated gold sputtered plastic film and electrode design of the invention

Figure IB shows the adhesive spacer with laser cut circular hole for overlaying on the gold sputtered plastic film

Figure 1C shows the assembled biochip electrochemical sensor of the invention
with the sensing reagent comprising antibody coated on the electrodes

Detailed Description of the Preferred Embodiments

The invention is a disposable electrochemical immunosensor of high sensitivity, and low cost. In one embodiment, the immunosensor of the invention is now described in detailed with reference to Figures 1A to 1C. Figure 1A shows the metal-sputtered polyester film substrate 101 in which are formed a reference electrode 111, a working electrode 112 and a counter electrode 113. The electrodes 111, 112 and 113 are delineated on the substrate 101 by forming breaks 114 in the coating. An adhesive film spacer 120 with an opening 121 as shown in Figure IB is affixed onto the substrate 101, to form the electrochemical immunosensor 100, as shown in Figure 1C. As shown in Figure 1C, the opening 121 in the adhesive spacer overlays the three electrodes to form the "well" of the electrochemical immunosensor, wherein the reagents are immobilized. The adhesive polymer spacer covers a major portion of the surface of the sensor, while the lower portion is left uncovered to provide electrical contact.

In one embodiment, the metal film of the immunosensor 100 and therefore the
material of the electrodes 111, 112 and 113 is gold- Alternatively, sputtered films of
platinum or palladium can also be used.

In one embodiment the immunosensor 100 is immobilized with a primary antibody
configured to bind with disease markers such as pathogens or their antigens within
human or animal tissue. Examples include various bacterial or viral agents causing
diseases such as anthrax, tuberculosis, salmonellosis, hepatitis, ebola etc.

In one embodiment the immunosensor 100 is immobilized with a primary antibody
configured to bind to cardiac disease markers such as cardiac troponin and CK-mb,
thyroid markers (TSH), or other disease markers including diabetic markers like
glucose, HblAC and insulin.

In one embodiment the immunosensor 100 is immobilized with a primary antibody
configured to bind with fungal pathogens or their antigens.

In one embodiment the immunosensor 100 is immobilized with a primary antibody
configured to bind with a toxic environmental contaminant or its antigen such as
pesticides, herbicides, mycotoxins.

In one embodiment the immunosensor 100 is used to carry out a sandwich assay.
In one embodiment the immunosensor 100 is used to carry out a competitive
assay.

In some embodiments the immunosensor 100 is further immobilized with a
secondary antibody such as alkaline phosphatase or glucose oxidase as labeling
reagent and horseradish peroxidase (HRP) as amplification agent.

In one embodiment the immunosensor 100 is immobilized with antibodies and
reagents functionalized within carbon nanotubes.

In one embodiment, the reference electrode 111 is coated with a redox mediator
such as potassium ferricyanide, or alternatively, hexamine ruthenium chloride in a

substrate reagent comprising glucose. In an alternative embodiment, the reference electrode can be coated with silver-silver chloride to provide a stable potential. The construction and operation of the electrochemical immunosensor of the invention is illustrated with an example.

Construction

The plastic biochip electrochemical immunosensor was constructed out of a polyester substrate of 250 u,m (10 mil) thickness with gold sputtered thickness of 300 nanometers and with a resistivity of < 3 ohms/cm procured from Technimet, USA. The electrode pattern, a 3-electrode system of custom design as shown in Figures 1A to 1C was delineated on the metal sputtered polyester film by laser ablation using a C02 laser system. A single sided adhesive spacer from Adhesives Research Inc, USA of 125 u.m (5 mil) thickness was laser cut with a circular hole. The laser cut adhesive spacer was overlaid on the laser ablated gold sputtered substrate to define the electrode areas precisely. The use of redox mediator (such as potassium ferricyanide) is essential in developing a pseudo-reference potential at the electrode surface that in turn provides a stable reference potential. This is different from the conventional silver-silver chloride reference electrode, gold working electrode and platinum counter electrode configuration. The so-called "well" created at the electrode areas by the circular cutout of the adhesive spacer can be used for immobilizing antibodies for any electrochemical assay.

Operation

The plastic biochip electrochemical immunosensor was fabricated by the process outlined above. The electrochemical sensor was cleaned with 10% methanol initially and then the complete electrode area was incubated with 10 u.g/ml of thiolated protein-A solution for 3 hours at 37°C After the incubation period, the sensor was washed with 10 mM PBS solution followed by rinsing with de-ionized water. A 10% skim milk reagent at 37°C for 1 hour was used to block the non-specific sites of the plastic electrochemical sensor covered with Protein-A layer. The capture antibody was then immobilized on the thiolated protein-A layer by incubating it for 1 hr at 37°C. The sensor was then washed, dried and packaged in ready to use condition for immunoassays.

For proceeding with the immunoassay, the sensor was further incubated with a sample containing the antigen of interest and then after washing, was further incubated with a secondary antibody labeled with glucose oxidase. The biochip sensor was then washed and the immunocomplex formed was exposed to a substrate solution comprising of 300 mg/dL glucose and 100 mM of potassium ferricyanide. Alternatively, hexamine ruthenium chloride can also be used as mediator. The electrochemical detection was carried out by chronoamperometry at 0.4 V using an electrochemical analyzer.
The disclosed immunosensor has nearly 3 times the sensitivity of conventional carbon screen printed electrodes, and therefore has fast response time. The sensitivity of the plastic biochip electrochemical immunosensor can be further enhanced by the use of carbon nanotubes functionalized with biomolecules.

The plastic biochip electrochemical immunosensor is reusable but its benefit lies in the fact that it costs few Indian rupees and can be used in single-use disposable clinical diagnosis and for monitoring environmental pollutants in bodily fluids, or biological systems.

We claim:

1. A disposable electrochemical immunosensor comprising: a polyester substrate with a sputtered metallic layer on one surface thereof; a sensor comprising a working electrode, a reference electrode and a counter electrode formed on the metallic layer; and a polymer film spacer comprising an opening, adhesively bonded onto a portion of the metallic layer such that the opening overlays the working electrode, reference electrode and counter electrode of the sensor, wherein the sensor electrodes on the metallic layer and the opening on the polymer film spacer are formed by laser ablation, and wherein a biological protein layer comprising antibodies modified to include thiol groups is immobilized within the opening on the polymer film spacer in contact with the electrodes.

2. The electrochemical immunosensor as recited in claim 1, wherein metallic layer is selected from one of gold or platinum or palladium.

3. The electrochemical immunosensor as recited in claim 2, wherein the reference electrode is electroplated with silver-silver chloride.

4. The electrochemical immunosensor as recited in claim 2, wherein one of the antibodies is labeled with one or more of alkaline phosphatase, horseradish peroxidase (HRP) or glucose oxidase.

5. The electrochemical immunosensor as recited in claim 4, wherein the detection is carried out using a substrate reagent comprising glucose and one of potassium ferricyanide or hexamine ruthenium chloride as mediator.

6. The electrochemical immunosensor of claim 2, wherein the sensitivity of the immunoassay is enhanced by use of carbon nanotubes deposited on the working electrode.

7. The electrochemical immunosensor as recited in claim 2, wherein a sandwich or competitive type immunoassay is performed by immobilizing antibodies for the target molecule.