# US 8,569,006 B2 · Cell-enzyme based biosensors

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- Number: US 8,569,006 B2
- Document ID: US8569006B2
- Status: Granted
- Kind: B2
- Application number: 12/919,725
- Filed: 2008-02-29
- Published: 2011-01-06
- Granted: 2013-10-29
- Earliest claimed: 2008-02-29
- Assignees: Imec; Katholieke Universiteit Leuven, KU LEUVEN R&D
- Applicants: Dries Braeken; Danielle Rand; Carmen Bartic
- Inventors: Dries Braeken; Danielle Rand; Carmen Bartic
- Web page: https://patentgrep.com/patent/US8569006B2

## CPC

- G01N 33/54366: {Apparatus specially adapted for solid-phase testing}
- G01N 33/5058: {Neurological cells}
- G01N 33/94: involving narcotics {or drugs or pharmaceuticals, neurotransmitters or associated receptors}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention relates to a sensor comprising a sensing layer and a surface layer, wherein said surface layer comprises, a first region suitable for adherent growth of cells, and a second region, adjacent to said second layer, suitable for the attachment of proteins, wherein the first and second region are in contact with the sensing layer.

## Claims

### Claim 1

A biosensor, the biosensor comprising:
a sensor configured to detect a change in a charge distribution;
a sensing layer, wherein the sensing layer is situated on the sensor; and
a surface layer, wherein the surface layer is situated on the sensing layer, and wherein the surface layer comprises a first region that is enzyme-free and adapted for adherent growth of cells and a second region, adjacent to the first region, wherein the second region is configured for attachment of proteins and comprises a bienzyme system, wherein the bienzyme system is adapted for conversion of a compound produced by the cells into a product which generates a signal detectable by the sensor, wherein the first region and the second region are separated from each other but in contact with the sensing layer, wherein at least one of the first region and the second region comprises a pattern of a plurality of linear elements, and wherein the first region and the second region are configured such that the cells row exclusively in the first region.

### Claim 2 (depends on 1)

The biosensor of claim 1, further comprising cells on the first region.

### Claim 3 (depends on 1)

The biosensor of claim 1, wherein at least one of the first region and the second region comprises a grid like pattern of a plurality of linear elements.

### Claim 4 (depends on 3)

The biosensor of claim 3, wherein the linear elements of the first region have a width of from about 5 micrometers to about 20 micrometers.

### Claim 5 (depends on 3)

The biosensor of claim 3, wherein the linear elements of the second region have a width of from about 2 micrometers to about 20 micrometers and a thickness of less than 0.5 micrometers.

### Claim 6 (depends on 1)

The biosensor of claim 1, wherein the cells are neuronal cells.

### Claim 7 (depends on 1)

The biosensor of claim 1, wherein the bienzyme system comprises L-glutamate pyruvate transaminase in combination with glutamate oxidase or glutamate dehydrogenase.

### Claim 8 (depends on 1)

The biosensor of claim 1 wherein the compound produced by the cells is a neurotransmitter.

### Claim 9 (depends on 8)

The biosensor of claim 8, wherein the neurotransmitter is glutamate.

### Claim 10

A method for detection of production of a compound by cells, the method comprising:
providing a biosensor comprising: a sensor configured to detect a change in a charge distribution; a sensing layer, wherein the sensing layer is situated on the sensor; and a surface layer, wherein the surface layer is situated on the sensing layer, and wherein the surface layer comprises a first region that is enzyme-free and adapted for adherent growth of cells and a second region, adjacent to the first region, wherein the second region is configured for attachment of proteins and comprises a bienzyme system, wherein the bienzyme system is adapted for conversion of a compound produced by the cells into a product which generates a signal detectable by the sensor, wherein the first region and the second region are separated from each other but in contact with the sensing layer, and wherein at least one of the first region and the second region comprises a pattern of a plurality of linear elements, and wherein the first region and the second region are configured such that the cells grow exclusively in the first region;
providing cells, wherein the cells undergo adherent growth on the first region of the sensor, whereby the compound is produced;
converting, by the bienzyme system of the second region, the compound into the product which generates a change in a charge distribution in the sensing layer; and
detecting the change in the charge distribution by the sensor of the biosensor, whereby production of the compound by the cells is detected.

### Claim 11 (depends on 10)

The method of claim 10, wherein the detectable signal is a change in pH.

### Claim 12 (depends on 10)

The method of claim 10, wherein the cells are neuronal cells and the compound is a neurotransmitter.

### Claim 13 (depends on 1)

The biosensor of claim 1, wherein the sensor is selected from the group consisting of a field effect transistor, a capacitive sensor, and a microelectrode.

### Claim 14 (depends on 13)

The biosensor of claim 13, wherein the sensor is a field effect transistor.

### Claim 15 (depends on 14)

The biosensor of claim 14, wherein the field effect transistor is an ion-selective field-effect transistor.

### Claim 16 (depends on 14)

The biosensor of claim 14, wherein the biosensor is configured such that the signal modifies a conductivity in a channel region of the field effect transistor.

### Claim 17 (depends on 7)

The biosensor of claim 7, wherein the second region comprises a pattern of longitudinal lines of glutamate oxidase or glutamate dehydrogenase and a pattern of traverse lines of L-glutamate-pyruvate-transaminase.

### Claim 18 (depends on 13)

The biosensor of claim 13, further comprising a reference electrode, wherein the sensor is a microelectrode comprising a pH sensitive layer, and wherein the biosensor is configured to measure a change voltage difference across the pH sensitive layer that arises from a change in pH or from a different charging of a double layer capacitance.

### Claim 19 (depends on 10)

The method of claim 10, wherein the sensor is a field effect transistor, and wherein a channel region of the field effect transistor is underneath the sensing layer.

### Claim 20 (depends on 19)

The method of claim 19, wherein the detectable signal modifies a conductivity in the channel region of the field effect transistor.

### Claim 21 (depends on 15)

The biosensor of claim 15, wherein the sensing layer is a pH sensitive gate layer, and wherein the biosensor is configured such that a change in charge distribution adsorbed at or created near the pH sensitive gate layer modulate a conductivity of a channel underneath.

### Claim 22 (depends on 1)

The biosensor of claim 1, wherein the bienzyme system is spotted at dedicated places, such that, in use, the product is locally concentrated.

### Claim 23 (depends on 1)

The biosensor of claim 1, wherein enzymes of the bienzyme system are immobilized in a ratio of from 100/1 to 1/100.

### Claim 24 (depends on 1)

The biosensor of claim 1, wherein enzymes of the bienzyme system are immobilized in a ratio of from 5/1 to 1/5.

### Claim 25 (depends on 1)

The biosensor of claim 1, further comprising a physical barrier configured to avoid growth of the cells outside of the first region.

### Claim 26 (depends on 1)

The biosensor of claim 1, further comprising a chemical barrier configured to avoid growth of the cells outside of the first region.