FIELD OF THE INVENTION

The present invention relates to microfluidic cartridge for biosensor and immunoassay applications.

BACKGROUND

Immunoassays and Bioassays are commonly performed today for the screening and detection of various disease markers or environmental pollutants. Typical assays require complex instrumentation which do calibration and perform reagent washing and are thus only limited to their use in laboratories. The advent of point-of-care device necessitates low cost disposable high performance microfluidic cartridge that has similar performance features as the traditional lab, however it is limited until now due to high manufacturing cost and heavy infrastructure requirements. In particular, biosensors and immunosensors based on electrochemical detection are simple and require washing steps to ensure reliability of the assay. These assays are simple and low cost and are gaining popularity for their use in point-of-care applications for the development of hand-held devices. One of the technology limitations for commercializing microfluidic cartridges is lack of a single use, low cost manufacturing process.

Current microfluidic cartridges require sophisticated molds to create micron size geometries and are very expensive and complicated (e.g. US7666285). Further integrating various components such as sensor chips, reagent reservoirs, micropumps, sampling port, waste chamber etc. is a challenge and often associated with low manufacturing yields. Thus there exists a need for a single use disposable, high performance microfluidic cartridge that uses low cost electrochemical sensors for biosensor and immunosensor applications.

Many immunosensor devices have been disclosed in the prior art. US patents 7682833 and 7723099 to Miller et al. disclose a disposable cartridge device with the microfluidic channels molded into the plastic top cover of the device. The device uses a combination of inbuilt dry reagents and fluids supplied by pumping, to carry out the diagnostics. US patent 7497997 to Glezer et al. discloses assay cartridges incorporating laminated sensors that are optically readable. However, the device is complicated and has to be fitted into an external reader for operating the pumps.

SUMMARY OF THE INVENTION

The invention is a microfluidic cartridge for detecting a molecule in an analyte, comprising a bottom cover, a transparent top cover, and a laminated microfluidic substrate therebetween. The laminated microfluidic substrate further comprises a polymer layer, a conductive sensor layer over the polymer substrate comprising at least one sensor, a double-sided adhesive layer over the polymer substrate and the conductive sensor layer comprising microfluidic channels laser cut thereon, and a transparent polymer layer overlaying the adhesive layer, that is laser cut to provide access to the conductive sensor layer at at least one location. The bottom cover is provided with a chamber to hold runoff fluid from the microfluidic substrate and an air vent to facilitate flow. The top and bottom covers are sealed along the outer edges.

The transparent top cover comprises at least two reservoirs to hold reagents, and at least three access ports including a sample input port for accessing the laminated microfluidic substrate, the cartridge further comprising a detachable overlay cover including micropumps to convey reagents from the at least two reservoirs through the at least three access ports to the laminated microfluidic substrate. The at least three ports for accessing the laminated microfluidic substrate of the transparent top cover are covered by a silicone septum for access to the micropumps.

The conductive sensor layer of the laminated microfluidic substrate comprises of a gold sputtered layer or a screen printed layer, and the polymer layer is a polyester film, the conductive sensor layer further including a laser inscribed pattern comprising at least one electrochemical sensor and a plurality of sensors for detecting presence of fluid.

The polyester film of the laminated microfluidic substrate is of at least 150 microns thickness and the gold sputtered layer is of at least 300 nm thickness with ohmic resistance of 3-4 ohms per sq. m.

The double-sided adhesive layer is of at least 250 microns thickness.

BRIEF DESCRIPTION OF THE DRAWINGS

A high performance microfluidic cartridge for biosensor/ immunosensor applications is disclosed with reference to the drawings in which:

Figure 1A is a view of the assembled plastic biochip based microfluidic cartridge

Figure IB shows an exploded view of the microfluidic cartridge delineating the component parts

Figure 2A is a view of another embodiment of the assembled microfluidic cartridge

Figure 2B shows the molded plastic top cover and bottom cover of the
microfluidic cartridge

Figure 2(c) shows the fabricated microfluidic cartridge in assembled condition

Figure 2(d) shows the fabricated microfluidic cartridge with syringe attached to sample port and micropumps connected to the top cover

Detailed Description

The present invention relates to the design and development of a novel, inexpensive, plastic microfluidic cartridge for biosensor and immunoassay applications. In the present invention, the plastic microfluidic cartridge is based on a plastic polyester substrate that serves dual purpose as a gold sensor and a laminated adhesive layer with top cover. This sensor layer which is based on gold sputtered sheet is laser ablated to define sensor areas. This sensor layer is further overlaid with adhesive spacer that is laser cut with a microfluidic geometry to define the fluidic channels for sample and reagent flow towards the sensor and to the waste collection area. It is a high performance cartridge as it has typical traditional laboratory measurement device features miniaturized into a cartridge format (Lab-on-Cartridge) and can be commercially manufactured at very low cost without any heavy infrastructure requirements for approximately INR TEN rupees in small quantities, thus enabling it to be used in single use disposable biosensor and immunosensor applications.

One embodiment of the high performance microfluidic cartridge of the invention is shown in Fig. 1A and IB. The assembled and sealed microfluidic cartridge 100 is shown in Fig. 1A, while an exploded view of the microfluidic cartridge delineating the various components is shown in Fig. 1B. In Fig. 1A, the cartridge 100 comprises of a top cover 101 with molded reagent reservoirs 103 and sampling port, 104 and a bottom cover 102. A laminated microfluidic substrate 110 comprising an electrochemical biosensor/immunosensor is placed between the top and bottom covers as shown in Fig. 1A. All the three parts, the top cover 101, laminated microfluidic substrate 110, and bottom cover 102 are ultrasonically bonded to yield an integrated, high performance microfluidic cartridge 100.

Further details of the device are shown in the exploded view of Fig. IB, wherein the laminated microfluidic substrate 110 is further separated into a polymer layer with a conductive coating 111, a double-sided adhesive layer 112 comprising microfluidic channels 113 and a transparent polymer layer 114 over the adhesive layer. Transparent polymer layer 114 is configured to be hydrophilic to ensure fluidic properties and the layers are laminated together to form the microfluidic substrate.

The conductive sensor layer 111 comprises biosensors or other sensors that are inscribed into the conductive portion by laser scribing or other suitable process. The microfluidic channels 113 on the adhesive layer 112 are also formed using laser cutting. The transparent hydrophilic polymer layer 114 is provided with one or more cutouts (not shown) to provide access to the microfluidic substrate.

Figure 1B also shows overlay cover 120 that is detachably affixed on to the top cover 101. Overlay cover 120 comprises at least three micropumps 121 configured to access the reagent reservoirs 103 and sample port 104.

Another embodiment of the invention is shown in Fig. 2A through 2E. As shown in
Fig. 2A, top cover 201 comprises reagent reservoirs 203 and sample input port 204. Figure 2B shows laminated microfluidic substrate 210, and microfluidic channels 213, while Fig. 2C shows bottom cover 202. Figure 2D shows the microfluidic cartridge device in assembled condition.

The device configured for testing is shown in Fig. 2E, with a sample introduced through sample input port 204 through syringe S. Micropumps 221 are shown externally connected to access the reservoirs 203 and sample port 204.

The laminated microfluidic substrate 110 or 210 may incorporate a conductive sensor layer 111 that is either gold sputtered or it may be of other conductive material such as carbon and produced by screen printing. In one embodiment the sensor layer 111 is polyester film of at least 150 microns thickness, and more preferably of 250 microns thickness. In one embodiment the thickness of the sputtered gold coating of the sensor layer 111 is 300 nm with ohmic resistance of 3-4 ohms per sq m. In one embodiment the double-sided adhesive layer 112 is preferably of at least 250 microns thickness, and more preferably of 400 to 500 microns thickness. The material of the adhesive layer 112 is preferably as available from 3M Adhesives.

Example

The microfluidic cartridge was fabricated as described above and shown in Fig. 2A through 2E for use with a point-of-care device called iSTATLAB. A typical breadboard sandwich Immunoassay using electrochemical detection was performed by employing high performance micropumps 221 that were integrated with the novel microfluidic cartridge 200 (as shown in Fig. 2D) to complete a lab-style assay with onboard reagent washing with air and liquid and delivery of electrochemical enzyme substrate using one of the micropumps to the electrochemical immunosensor. Wash reagent comprising of PBS Tween buffer was stored in one of the reservoirs, while the other reservoir had Napthyl Phosphate, an electrochemical substrate for the alkaline phosphatase enzyme based Immunoassay. Antigen samples for assay were delivered to the electrochemical immunosensor via a syringe S attached to the sample port 204 on the microfluidic cartridge 200, as shown in Fig. 2E and the assay was carried out.

This novel low cost microfluidic cartridge with on-board reagent washing feature and in-built calibration feature can be used with biosensors and/or immunosensors in few microliters of sample using any of the known electrochemical techniques to give laboratory like superior performance to the point-of-care device. The microfluidic cartridge enables for the first time the use of biosensor and immunosensor chips for diagnostic applications commercially in a single use disposable format at a very low cost. The plastic microfluidic device is highly versatile as it can be used for any other electrochemical biosensor application where lab like performance at very low cost is required.

The plastic microfluidic cartridge of the invention can also be used for other immunoassays such as

1. Cardiac Markers (Cardiac Troponin, CK-mb)
2. Thyroid Markers (TSH)
3. Disease markers (diabetic markers like Glucose, Hb1AC, Insulin)
4. Environmental pollutants (pesticides, herbicides, mycotoxins etc)

The inventive device configuration can also be applied in other biosensor applications such as measurement of Glucose, HbA1C and in the measurement of electrolytes and blood gases with improved accuracy, compared to other equipment presently in use.

I claim:

1. A microfluidic cartridge for detecting a molecule in an analyte, comprising:

a bottom cover, a transparent top cover, and

a laminated microfluidic substrate therebetween; the laminated microfluidic substrate comprising:

a polymer layer;

a conductive sensor layer over at least a portion of the polymer substrate
comprising at least one sensor;

a double-sided adhesive layer over the polymer substrate and the conductive sensor layer comprising microfluidic channels laser cut thereon; and

a transparent polymer layer overlaying the adhesive layer, that is laser cut to provide access to the conductive sensor layer at at least one location;

the bottom cover comprising a chamber to hold runoff fluid from the
microfluidic substrate, and an air vent;

the transparent top cover comprising at least two reservoirs to hold reagents, and at least three ports including a sample input port for accessing the laminated microfluidic substrate, wherein,

the top and bottom covers of the cartridge are sealed along the outer edges, the cartridge further comprising a detachable overlay cover including micropumps to convey reagents from the at least two reservoirs through the at least three access ports to the laminated microfluidic substrate.

2. The microfluidic cartridge as recited in claim 1, wherein the at least two ports for accessing the laminated microfluidic substrate of the transparent top cover are covered by a silicone septum for access to the micropumps.

3. The microfluidic cartridge as recited in claim 1, wherein the conductive sensor layer comprises of a gold sputtered layer or a screen printed layer.

4. The microfluidic cartridge as recited in claim 1, wherein, the polymer layer is a polyester film and the conductive sensor layer is a gold sputtered layer, the gold sputtered layer further including a laser inscribed pattern comprising at least one electrochemical sensor and a plurality of sensors for detecting presence of fluid.

5. The microfluidic cartridge as recited in claim 4, wherein the polyester film is of at least 150 microns thickness and the gold sputtered layer is of at least 300 nm thickness with ohmic resistance of 3-4 ohms per sq. m.

6. The microfluidic cartridge as recited in claim 1, wherein the thickness of the double-sided adhesive layer is at least 250 microns.

7. A microfluidic cartridge for detecting a molecule in an analyte, comprising:

a bottom cover, a transparent top cover, and

a laminated microfluidic substrate there between;

the laminated microfluidic substrate comprising:

a polymer layer;

a conductive sensor layer over at least a portion of the polymer substrate
comprising at least one sensor;

a double-sided adhesive layer over the polymer substrate and the conductive sensor layer comprising microfluidic channels laser cut thereon; and

a transparent polymer layer overlaying the adhesive layer that is laser cut to provide access to the conductive sensor layer at at least one location;

the bottom cover comprising a chamber to hold waste fluid from the microfluidic substrate and incorporating an air vent;

the transparent top cover comprising at least two reservoirs to hold reagents, and at least three ports including a sample input port, for accessing the laminated microfluidic substrate; wherein,

the top and bottom covers are sealed along the outer edges.

8. The microfluidic cartridge recited in claim 7, further comprising micropumps to convey reagents from the at least two reservoirs through the at least three access ports to the laminated microfluidic substrate, wherein the micropumps are located externally of the device.

9. A method of detecting a biological molecule using the microfluidic cartridge recited in claim 1 or claim 7.