WO2015007369A1

WO2015007369A1 - Biosensor array

Info

Publication number: WO2015007369A1
Application number: PCT/EP2014/001804
Authority: WO
Inventors: Masaki Hasegawa
Original assignee: Merck Patent GmbH
Current assignee: Merck Patent GmbH
Priority date: 2013-07-19
Filing date: 2014-07-01
Publication date: 2015-01-22
Anticipated expiration: 2016-01-19

Abstract

The invention relates to a biosensor array and to preparation thereof. The invention further relates to use of biosensor cells specifically in biosensor arrays. The invention further relates to a calibration method of a biosensor array and a method for wider and improved dynamic detection range of a biosensor array.

Description

Biosensor array

Field of the Invention

The invention relates to a biosensor array and to a preparation thereof. The invention further relates to the use of biosensor cells specifically in biosensor arrays. The invention further relates to a calibration method of a biosensor array and a method for wider and improved dynamic detection range of a biosensor array.

Background and Prior Arts Biosensor array configurations are already described, for example, in JP3969702B, JP4255475B, and WO2007/084077A.

JP3969702B (Seiko Epson Co.) describes a capacitor type TFT biosensor array.

JP4255475B (Fujitsu Ltd.) describes a top gate type TFT biosensor array.

WO 2012/084077A (Agency for Science, Technology and Research) describes an FET biosensor array.

However, there are still one or more considerable problems for which improvement is desired, as listed below.

1. Dynamic detection range should be wider and improved.

2. There is still a need for improvement in the sensitivity of the biosensor. Detailed Description of the Invention

The inventors aimed to solve one or more of the aforementioned problems. Surprisingly, the inventors have found an inventive biosensor array comprising a plurality of biosensor cells (4) having a sensor layer (14) and receptors (13) that are immobilized on surface of the sensor layer (14), where at least one biosensor cell of the plurality of biosensor cells has a different density of receptors than the other biosensor cell of the plurality of biosensor cells. The inventive biosensor array is highly suitable for detecting bioanalytes. Preferably, it solves one or both of problems 1 and 2.

The inventors further innovated a calibration function of a biosensor array for solving problem 1 or both of problems 1 and 2.

The invention therefore relates to a biosensor array which comprises:

• a substrate (8), comprising switching field effect transistors (3), gate lines (2), drain lines (5), and source lines (1);

• a plurality of biosensor cells (4), which are connected to the source lines (1) via switching field effect transistors (3); where each of the biosensor cells has a source electrode (11), a drain electrode (12), a sensor layer (14) and receptors (13), which are immobilized on the surface of the sensor layer (14); with the proviso that at least one biosensor cell of the plurality of biosensor cells has a different density of receptors than the other biosensor cell of the plurality of biosensor cells and a differentiation of the receptor density between at least one biosensor cell of the plurality of biosensor cells and the other biosensor cell of the plurality of biosensor cells is in the range of 1.05 times to 1000 times. Preferably, differentiation of the receptor density between at least one biosensor cell of the plurality of biosensor cells and the other biosensor cell of the plurality of biosensor cells is in the range of 2 times to 15 times.

Wider and improved dynamic detection range can be realized by

aforementioned biosensor array of the present invention. More specifically, by switching from biosensor cells with high sensitivity to biosensor cells with low sensitivity, wider and improved dynamic detection range can be realized. The switching from biosensor cells with high sensitivity to biosensor cells with low sensitivity can be realized by selecting switching field effect transistors (hereafter, SWFETs) accordingly.

One or more of the biosensor cells of the plurality of the biosensor cells with lower receptor density than the other biosensor cell of the plurality of the biosensor cells is used as the low sensitivity biosensor cells, and the biosensor cells with higher receptor density are used as the high sensitivity biosensor cells.

Preferably, the biosensor cells of the present invention are, at each occurrence, field effect transistors to realize higher sensitivity of the biosensor array. The SWFET may or may not be present in the biosensor array. In case the SWFETs are not present, the biosensor cells function instead as SWFETs.

The configuration, in which the biosensor cells function instead as

SWFETs, makes the structure of the biosensor array simple and may realize cost reduction of the biosensor array.

The biosensor cells in the biosensor array may have, at each occurrence, a top gate electrode and / or a bottom gate electrode.

Preferably, the biosensor cells in the biosensor array of the present invention have a top gate electrode. A sensor layer of the biosensor cell is not particularly limited. Many kinds of semiconductive materials which are publicly known, can be used as materials for the sensor layer. Such as amorphous silicon, polycrystalline silicon, metal oxide material, organic semiconductor material, CNT, graphene, reduced graphene oxide (hereafter, RGO).

Graphene or RGO is preferable because it is highly suitable to fix linkers and / or receptors on the surface.

The sensor layer may be a stacked layer to enhance the electron mobility of the sensor layer.

The stacked layer consists of two or more of layers.

As a preference, the stacked layer consists of two layers and one layer is a RGO layer. The RGO layer is stacked onto the other sensor layer that has preferably a higher electron mobility than the RGO layer. The other sensor layer of the stacked layer comprises polycrystalline silicon, or metal oxide material to increase the electron mobility of the stacked layer.

As a preference, the biosensor array according to the present invention comprises a biosensor array area and one or more calibration cell areas. The calibration cell area can be overlapping with the biosensor array area and / or can be located in a different part of the biosensor array area.

Preferably this configuration can be used in the calibration process.

All calibration process can be done in the calibration area, and the actual biosensing to detect analyte type and analyte concentration can be done in the biosensor array area.

A substrate of the biosensor array is not particularly limited. Any publicly known kind of substrates having good surface flatness can be used, such as a glass substrate, a silicon substrate, or a metal substrate. Preferably, the biosensor array may comprise a wall to keep a buffer solution. A material for the wall is not particularly limited. It can be selected from publicly known ones, such as silicone rubber gel.

As a preference, at least one of the biosensor cells in the biosensor array according to the present invention, consists of different kinds of receptors compared with the receptors of the rest of the biosensor cells of the biosensor array, to detect different kinds of analytes.

By immobilizing different kind of receptor from one or more of the rest of biosensor cells of the biosensor array, the biosensor array of the present invention can have the ability to detect multiple analytes by one biosensor array at the same time.

The kind of receptor is not particularly limited. Depending on the kind of target analyte, the receptor is selected preferably from publicly known ones.

Publicly known antibodies, aptamers or capture DNA can be used as a receptor of the present invention.

The buffer solution to disperse receptors is not particularly limited.

Depending on the kind of receptor, the solution can be preferably selected from publicly known buffer solutions for biotechnology. Such as PBS, HEPES buffer, Tris buffer. Preferably, NaCI is added to the solution to disperse the receptors.

Preferably, publicly known linkers can be used to fix receptors on the sensor layer stably. For example, sulfo-SSMCC cross linker, 1- pyrenebutanoic acid succinimidyl ester. The solvent for linker is not particularly limited. Publicly known one can be selected to make a linker solution.

For the purpose of the invention, DMF, Methanol can be used preferably as the solvents for aforementioned linkers.

The invention further relates to a process for the preparation of a biosensor array of the present invention, comprising the following steps in the sequence: a) performing film formation of sensor layer;

b) carrying out controlling receptor concentration dispersed in a buffer solution;

c) pouring process to place the buffer solution on the sensor layer;

d) incubation process to immobilize receptors on the sensor layer;

e) rinse process to wash out the buffer solution and receptors which did not immobilize on the sensor layer.

A receptor density of a biosensor cell can be controlled by changing the receptor concentration in the receptor solution and the incubation time.

The term "receptor density" is used with the meaning, that the receptor number / μιτι² is confirmed by the following method: The actual receptor density of biosensor cells is confirmed by Atomic force microscopy (here after, AFM), such as SPM-9700 (Shimazu) with eyes or with software system of SPM-9700 named" MODEL Particle Analysis" (Shimazu) for particle analysis. The AFM image with 300 nm² to 500 nm² resolution can be used to count a receptor density by eyes or by the software system. The results of AFM can be used to predetermine the receptor

concentration in a receptor solution and the incubation time in step d).

Preferably, the incubation time is 1 hour up to 15 hours. A rinse solution is not particularly limited and may be selected from publicly known ones. Preferably PH7 PBS solution can be used in the rinse process in step e).

Preferably, the process for the preparation of a biosensor array of the present invention embraces following sequential steps f) to j) between step a) and step b);

f) preparing a linker solution;

g) pouring process to place the linker on the sensor layer;

h) incubation process to fix the liner on the sensor layer;

i) rinse process to wash out the solution and linkers which did not immobilize on the sensor layer;

j) additionally, a drying process to remove the rinse solution from the substrate may be present.

Preferably, in step f), a buffer for a linker is saturated with a linker to make a linker solution.

As a preference, incubation condition in step h) is 1 to 10 hours settlement at room temperature in air conditioning. More preferably, it is 4 hours settlement at room temperature in air conditioning.

A rinse solution is not particularly limited and may be selected from publicly known ones.

Preferably PH7 methanol / PBS mixed solution can be used in the rinse process in step i).

As a preferred embodiment of the dry process in step j), a biosensor array is settled in air conditioning for 1hour at room temperature.

Preferably, an inkjet machine, a dispenser or a nozzle printing machine can be used to pour a buffer solution selectively on a sensor layer of the biosensor cells. An inkjet machine, a dispenser, or a nozzle printing machine can pour different solutions which have a different concentration of receptors and / or different kinds of receptor, selectively on different biosensor layers.

Mask technique is also applicable to pour a buffer solution selectively on a sensor layer of the biosensor cells.

Rubber masks, preferably a silicone rubber mask, can be used in this way. In case the mask technique is used in the process for the preparation of a biosensor array of the present invention, the process has the following sequential steps a), b) and k) to o), instead of present sequential steps of the process for the preparation of a biosensor array of the present invention a) to e); a) performing film formation of sensor layer;

k) a rubber mask is put onto a biosensor array to cover the sensor layers of one or more of biosensor cells of the plurality of biosensor cells selectively.

Then, I) a solution having determined concentration of receptor is poured onto one or more of the biosensor layers of a biosensor array, m) the mask is removed and put again onto a biosensor array to cover the sensor layers of another biosensor cells of the plurality of biosensor cells selectively. Then n) a solution having different concentration or different kind of receptor is poured onto the biosensor layers. And then, o) the mask is removed again.

Repeating aforementioned process, the biosensor cells having different receptor density and / or different kinds of receptors can be fabricated. The receptor density of a biosensor cell can be controlled by changing receptor concentration in a receptor solution and / or incubation time. The invention further relates to the use of biosensor cells specifically in a biosensor array of the present invention, in which the biosensor array comprises:

• a plurality of biosensor cells (4), which are connected to the source lines (1) via switching field effect transistors (3); where each of the biosensor cells has a source electrode (11), a drain electrode (12), a sensor layer (14) and receptors (13), which are immobilized on the surface of the sensor layer (14); with the proviso that at least one biosensor cell of the plurality of biosensor cells has a different density of receptors than the other biosensor cell of the plurality of biosensor cells and a differentiation of the receptor density between at least one biosensor cell of the plurality of biosensor cells and the other biosensor cell of the plurality of biosensor cells is in the range of 1.05 times to 1000 times.

The invention further relates to a calibration method of a biosensor array. According to the present invention, the term "calibration" is used with the meaning that p) making an estimate of the symbol K and A in the formula (I), and q) using the obtained values for K and A to conclude from the drain current to the analyte type and the analyte concentration. The values of the symbol K and A depend on an analyte type, receptor type and receptor density.

Drain current = K * Y + A Formula (I)

Where in formula (I), the symbol K depends on the analyte type, receptor type and receptor density . The symbol K represents the gradient of the calibration curves.

The symbol A represents the initial drain current of each biosensor cell of the biosensor array before any analyte solution is poured.

And the symbol Y represents an analyte concentration of the poured analyte solution.

When actual biosensing is carried out by using the biosensor array, calibrated analyte concentration and the analyte type are determined by measured drain current and the values of K and A obtained in the calibration.

The term "drain current change " in the formula (I) means a value calculated as follows: detected drain current - A.

For the purpose of this invention, the term "drain current" is used with the meaning of the raw drain current value of a biosensor array.

The calibration method embraces determination process of calibration curves of each biosensor cell. The determination process of calibration curves embraces following sequential process r) to w);

r) preparation process of two or more solutions that each independently have the same kind of an analyte with different concentration, such as 100 pico Mol / 10mM buffer , 1 nano Mol / 10 mM buffer, 10 nano Mol / 10 mM buffer, 100 nano Mol / 10 mM buffer, 1 micro Mol / 10 mM buffer of analyte concentration.

s) Measurement process of the value of the drain current to set as the value of A

t) pouring process of the solution with the lowest analyte concentration selected from the prepared solutions, to one or more of the biosensor cells;

u) measurement process of drain current;

v) pouring process of a solution which contains analyte with higher

concentration than the solution in step b); w) measurement process of drain current change.

Measurement process of drain current and drain current change may be continuous throughout the step t) to v) and also pouring process may be continuous by changing solutions with different concentration of analyte.

Measurement method and equipment are not particularly limited. Publicly known techniques and / or equipment can be used, such as digital signal oscilloscope with probe(s), or a digital altimeter.

Preferably a biosensor array of the present invention may have one or more of publicly known amplification circuits to make the drain current larger.

After in the step w), the following step x) preferably follows:

x) Plotting process of the measured value of the drain current change to determine the calibration curve of the biosensor cells

In each biosensor cells having different receptor condition, such as kind of receptor, density of receptor, the calibration method is applied to

determine the calibration curve of the biosensor cells and to confirm a relation between concentration of an analyte and drain current of the biosensor cells.

Primary approximation lines as calibration curves of biosensor cells of the biosensor array are calculated with the values of the first three or four drain current changes.

The two proportional coefficients with its value A and K of the formula (I) are determined from the primary approximation lines on a receptor type and a receptor concentration basis as well as a analyte type basis by each biosensor cells of the biosensor array. Determined calibration curves may be stored in a memory or an external storage on a receptor and an analyte type basis. As an external storage, preferably a laptop computer or a desktop computer may be used.

The biosensor array of the present invention may have a memory to store determined calibration curves and / or the memory may be an external memory.

The determined values of the symbols K and A in the earlier mentioned formula (I) may be stored in the memory or the external storage as mentioned above on a receptor type and receptor concentration basis as well as an analyte type basis.

When actual biosensing is carried out with the biosensor array, calibrated analyte concentration and the analyte type are determined by measured drain current and the values of K and A obtained in the calibration.

A memory and the external storage that has a calibration data, calibration curves, the values of the symbols K and A, may be used for the calibration of another biosensor cell and / or biosensor array.

Preferably, the stored calibration data may be used for the same biosensor array that was used in calibration process due to the products quality varies widely caused by variation of fabrication conditions.

The invention further relates to a method of wider and improved dynamic detection range and high sensitivity of a biosensor array.

To realise wider and improved dynamic detection range and high sensitivity of a biosensor array efficiently, the biosensor array has at least two types of biosensor cells, one is a biosensor cell with higher receptor density and another one is a biosensor cell with lower receptor density than the higher one. The biosensor cell with low receptor density is used to low sensitivity biosensor and the biosensor cell with high receptor density is used as high sensitivity biosensor.

By switching from biosensor cells with high sensitivity to biosensor cells with low sensitivity, wide detection range and high sensitive biosensor array can be realised.

For switching, the value of the saturation point of each biosensor cell can be determined as follows;

y) Pour at least six or more analyte solutions having different

concentration such as 100 pico Mol / 10 mM buffer, 1 nano, 10 nano, 100 nano, 1 micro, 0 micro, 20 micro, 30 micro and 50micro Mol / 10 mM buffer and measure drain currents of each biosensor cell of the biosensor array.

Then, z) plot relations between analyte concentrations and drain current changes. As a next step, A) calculate 1^st primary approximation line as calibration curve by using first three or four values of drain current changes.

Furthermore, B) by using the value of the last three drain current changes, 2^nd primary approximation line is determined. As a next step, C) extend both 1^st and 2^nd primary approximation lines to figure out the crossing point. The crossing point can be defined as the saturation point.

The value of the saturation point of each biosensor cell of the biosensor array may be stored in a memory or an external storage.

D) The determined value of the saturation point of each biosensor cell of the biosensor array is used to define the switching point of each biosensor cell.

In an actual biosensing, when the drain current exceeds the saturated point referred to the calibration curve, the SWFETs of the biosensor cells with higher sensitivity turn off, and the SWFETs of biosensor cells with lower sensitivity turn on to change biosensing sensitivity.

Definition of Terms

The term "field effect transistor" means a transistor in which the current path from source to drain is modulated by applying a transverse electric field between grid or gate electrodes. The term embraces thin film transistor (hereafter, TFT) and Metal Oxide Semiconductor Field Effect Transistor (hereafter, MOSFET).

Each feature disclosed in this specification, unless stated otherwise, may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless stated otherwise, each feature disclosed is but one example of a generic series of equivalent or similar features.

The invention is described in more detail in reference to the following examples, which are only illustrative and do not limit the scope of the invention.

Examples

Example 1 : Biosensor array fabrication

Amorphous Si:H (hereafter a-Si:H) thin film transistor (hereafter TFT) arrays are fabricated by using conventional plasma CVD and

photolithography method on one inch square silicon substrate as switching TFT.

The array consists of 2 rows and 2 columns, and the source electrodes made of gold are connected to each switching TFT. The drain electrodes made of gold are connected to each drain line. The source and drain electrodes are fabricated by using lift off deposition method.

The source and drain electrodes pattern is made with photoresist by photolithography, then Au is fabricated as the source and drain electrodes by using vapour deposition method. After Au is deposited, photoresist pattern is removed by solvent.

Instead of Au vapor deposition method, ink jet printing method can be used to fabricate Au pattern.

Then, the source and drain electrodes are coated by graphene oxide (hereafter, GO) that is dispersed in methanol, by using an inkjet printer.

Here, the GO is prepared as follows:

At first, graphite is mixed with the strong acid solution comprising NaNO3, H2SO4, and KMnO₄. Then the mixture is stirred for 50 hours. After stirring, the mixture is diluted in pure water, and it is centrifuged to separate exfoliated graphite. Then it is decanted off and dispersed in methanol.

After the GO is coated on the source and drain electrodes as the sensor layer, methanol is evaporated at room temperature, then the GO is reduced by ascorbic acid to a RGO, and RGO works as sensor layer. After reduction has been obtained, the silicone rubber mask is placed on the biosensor array to keep the solutions.

Then, the linker solution that consists of 1-pyrenebutanoic acid

succinimidyl ester and methanol is poured onto the RGO surface.

Methanol as a buffer solution of the linker solution is saturated with 1- pyrenebutanoic acid succinimidyl ester to make the linker solution. The pouring condition is 4 hours settle at room temperature to fix the linker on the RGO surface.

PH7 methanol / PBS mixed solution is used to rinse. After 1 hour settled, the biosensor array having 2 * 2 biosensor cells with linker is obtained. Then, IgE aptamer solution is poured onto one biosensor cell surface to fix the IgE aptamer on the linker as the receptor of the biosensor array.

The four types of IgE aptamer solutions are prepared with following conditions; 10 nM, 30 nM ,50 nM and 100 nM IgE aptamer, as the receptor, are dispersed each independently in the buffer solution.

As the buffer solution, 10 mM PBS with 140mM NaCI is used.

The four types of IgE aptamer solutions are poured each independently onto the four biosensor cells of the biosensor array.

After one hour incubation at room temperature, the biosensor cells are rinsed by PH7 PBS solution to wash out the buffer solution of the receptor and receptors which do not immobilize on the sensor layer.

Finally the biosensor array having 2 * 2 biosensor cells is obtained.

Receptor density of each biosensor cell of the biosensor array is 3000 / m², 4800 / prn² , 9000 / Mm² and 17,000/ μηι² confirmed by AFM, and the AFM image with 300 nm² resolution is used to count the receptor density by eyes.

Example 2: Determination of receptor density of the biosensor array

The AFM image with 300 nm² resolution is used to count the receptor density of the biosensor cells of the biosensor array of example 1. It is counted by eyes.

Receptor density of each biosensor cell of the biosensor array fabricated in the example 1 is 3000 / μπι², 4800 / μηη² , 9000 / m² and 17,000/ μηι². The measurement results of the biosensor cells of the biosensor array fabricated in the example 1 and relation between receptor concentration and receptor density in the fabrication condition in example 1 are shown in figure 12.

Example 3: Calibration

Analyte solutions with 1 pM, 5 nM, 20 nM, 25nM, 130 nM, 220nM, 500 nM and 1 μΜ IgE / 10 mM buffer, are prepared. As the buffer solution, 10mM PBS with 140mM NaCI is used. The biosensor array fabricated in the Example 1 is used in the calibration process.

Before pouring the analyte solutions, drain current of the biosensor cells are each independently measured with the digital signal oscilloscope Agilent DSO3202A is used to determine the value A of each biosensor cell of the biosensor array.

Then, analyte solutions, 1 pM, 5 nM, 20 nM, 25nM, 130 nM, 220nM, 500 nM and 1 μΜ IgE / 10 mM buffer with 10mM PBS and 140mM NaCI are poured according to the analyte concentration of the solutions and the drain current changes of the biosensor cell of the biosensor array are measured sequentially with the digital signal oscilloscope named Agilent DSO3202A.

The obtained data is plotted.

The primary approximation lines are calculated with the values of the first three drain current changes.

The two proportional coefficients with its value A and K of the formula (I) are determined from the primary approximation lines by each biosensor cells of the biosensor array.

Fig. 13 shows the results.

In fig. 13, diamond shapes represent drain current changes of the biosensor cell having 17,000/ μητι² IgE receptor density, square shapes represent drain current changes of the biosensor cell having 9,000/ μιτι² IgE receptor density, triangle shapes represent drain current changes of the biosensor cell having 4,800/ μιτι² IgE receptor density, and cross shapes represent drain current changes of the biosensor cell having 3,000/ μιτι² IgE receptor density.

Example 4: Specificity check of the antibody detection of the receptor IgE antibody solutions with 10 nM, 100 nM, and 200 nM IgE / 10 mM buffer with 10mM PBS and 140mM NaCI are prepared each independently. Furthermore BSA antibody solutions with 10 nM, 100 nM, and 200 nM BSA / 10 mM buffer with 10mM PBS and 140mM NaCI are prepared each independently.

Those antibody solutions are poured onto the biosensor cell having 17,000/ pm² IgE receptor density, which has IgE receptor on the sensor surface, of the biosensor array fabricated in the example 1.

Drain current changes are observed when the IgE antibody solutions are poured. On the other hand, the drain current change does not occur when the BSA antibody solutions are poured.

The results show the selectivity of receptor against target antibody.

And by changing the receptor type, on the sensor layer of the biosensor array, the biosensor cell specificity can detect antibody.

The results prove that where a receptor of at least one biosensor cell in the biosensor array consists of a different kind of receptor compared with receptors of the rest of the biosensor cells, the biosensor array has multiple detection function of antibodies.

Fig.14: shows the drain current change of the biosensor array fabricated in the example 1.

Description of the drawings

The invention is explained in greater detail below with reference to illustrative embodiments:

Fig. 1 : shows a biosensor array according to the present invention

Fig. 2: shows a biosensor array according to the present invention, in which the biosensor cells have a gate electrode and function as FETs. Fig. 3: shows a biosensor array according to the present invention, in which the switching FETs are not present and instead, the biosensor cells function as FETs.

Fig. 4: shows a biosensor array according to the present invention, in which the biosensor array area and the calibration cell area are located in different parts of the biosensor array.

Fig. 5: shows a biosensor array according to the present invention, characterized that the calibration cell area overlaps with the biosensor array area.

Fig. 6: shows a biosensor cell structure according to the present invention that does not have a gate electrode.

Fig. 7: shows a biosensor cell structure according to the present invention that comprises a reference electrode as a top gate electrode .

Fig. 8: shows a biosensor cell structure according to the present invention that comprises a top gate electrode.

Fig. 9: shows the A - A' cross section of the biosensor cell according to the present invention disclosed in Fig. 8.

Fig. 10: shows the B - B' cross section of the biosensor cell according to the present invention disclosed in Fig.8.

Fig. 11 : shows a biosensor cell structure according to the present invention that comprises a bottom gate electrode.

Fig. 12: shows the relation between receptor concentration and receptor density in the condition of working example 3. Fig. 13: shows the calibration curves of the biosensor array in working example 4.

Fiq.14: shows the drain current changes of the biosensor array in working example 4.

List of reference signs in figures

1. Source line

2. Gate line

3. Switching TFT

. Biosensor cell

5. Drain line

6. Calibration cell area

. Biosensor array area

8. Substrate

9. Wall

10. Insulator

11. Source electrode

12. Drain electrode

13. Receptor

4. Sensor layer

5. Buffer solution

6. Top gate electrode

7. Gate insulator

8. Bottom gate electrode

Claims

Patent Claims

1. A biosensor array which comprises:

2. The biosensor array according to Claim 1 ; where the differentiation of the receptor density between at least one biosensor cell of the plurality of biosensor cells and the other biosensor cell of the plurality of biosensor cells is in the range of 2 times to 5 times.

3. The biosensor array according to Claim 1 or Claim 2;

characterized in that the biosensor cells (4) are, at each occurrence, field effect transistors.

4. The biosensor array according to one or more of Claims 1 to 3,

characterized in that the switching field effect transistors (3) are not present and instead, the biosensor cells (4) function as field effect transistors.

5. The biosensor array according to one or more of Claims 1 to 4, characterized in that the biosensor cells (4) comprise at each occurrence at least one top gate electrode (16).

6. The biosensor array according to one or more of Claims 1 to 4, characterized in that the biosensor cells (4) comprise at each occurrence at least one bottom gate electrode (18) and at least one gate insulator (17).

7. The biosensor array according to one or more of Claims 1 to 6; characterized in that the sensor layer (14) of the biosensor cells (4) of the biosensor array comprises graphene.

8. The biosensor array according to one or more of claims 1 to 7; where the sensor layer of the biosensor cells (4) of the biosensor array comprises a reduced graphene oxide.

9. The biosensor array according to one or more of claims 1 to 8, where the biosensor array comprises a biosensor array area (7) and a calibration cell area (6).

10. The biosensor array according to one or more of claims 1 to 8, where the biosensor cells (4) comprise a layer of insulator (10) on at least a part of the drain electrode (12) and / or the source electrode (11).

11. The biosensor array according to one or more of claims 1 to 10, where the biosensor cells (4) comprise walls (9) to contain a buffer solution (15) .

12. The biosensor array according to one or more of claims 1 to 11 , where at least one biosensor cell of the plurality of biosensors has a different kind of receptors (13) than the other biosensor cells of the plurality of biosensor cells.

13. Use of biosensor cells in a biosensor array;

where the biosensor array comprises,

• a plurality of biosensor cells (4), which are connected to the source lines (1) via switching field effect transistors (3); where each of the biosensor cells has a source electrode ( 1), a drain electrode (12), a sensor layer (14) and receptors (13), which are immobilized on the surface of the sensor layer (14); with the proviso that at least one biosensor cell of the plurality of biosensor cells has a different density of receptors than the other biosensor cell of the plurality of biosensor cells and a differentiation of the receptor density between at least one biosensor cell of the plurality of biosensor cells and the other biosensor cell of the plurality of biosensor cells is in the range of 1.05 times to 1000 times.

14. Method for preparing biosensor cells of the biosensor array

according to one or more of Claims 1 to 12, comprising the following sequential steps;

a) perform film formation of the sensor layer;

b) prepare a buffer solution and control the receptor concentration in the buffer solution; c) pour the buffer solution onto the sensor layer;

d) incubate to immobilize the receptors on the sensor layer;

e) rinse to wash out the buffer solution and receptors which did not immobilize on the sensor layer;

here, the step b) may exist before the step a).

15. Method for preparing biosensor cells of the biosensor array

according to Claim 14,

where the method embraces step f) to i) sequentially between step a) and step b) in the following sequence;

f) preparing a linker solution;

g) pour the linker solution onto the sensor layer;

h) incubate to immobilize the liner on the sensor layer;

i) rinse to wash out the solution and linkers which did not immobilize on the sensor layer.

16. Method for preparing biosensor cells of the biosensor array

according to Claim 15,

where the method embraces the step j) after the step i), where step j) is a drying process.

17. Method for preparing biosensor cells of the biosensor array

according to one or more of Claims 14 to 16,

where the method contains the following sequential steps k) to o) instead of steps c) to e);

k) put a rubber mask onto a biosensor array to cover the sensor layers selectively;

I) pour a solution having a determined concentration of receptor onto one or more of the biosensor cells of a biosensor array;

m) remove the rubber mask after step d) or step e), and put the rubber mask again onto a biosensor array to cover different biosensor cells selectively. n) pour a solution having different concentration or different kind of receptor onto one or more of biosensor cells

o) remove the rubber mask.

18. A calibration method of a biosensor array according to one or more of Claims 1 to 12,

where the calibration method comprises the following sequential steps p) and q);

p) estimate the symbol K and A of the formula (I);

q) use the obtained values for K and A to conclude from the drain current to the analyte type and analyte concentration.

Drain current = K * Y + A Formula (I) where in formula (I), the symbol K represents the gradient of the calibration curves, the symbol A represents initial drain current before any analyte solution is poured, and the symbol Y represents an analyte concentration of a poured analyte solution.

19. The calibration method of a biosensor array accoring to Claim 18, where the step p) embraces the following sequential steps r) to w), and the step x) after the step w); r) prepare two or more of solutions that each independently have the same kind of an analyte with different concentration

s) measure the value of a drain current, and set as a value of A t) pour the solution with the lowest analyte concentration selected from the prepared solutions, to one or more of biosensor cells; u) measure the value of a drain current;

v) pour the solution which contains analyte with higher concentration than the solution in step s);

w) measure the value of a drain current; x) plot the measured value of the drain current change to determine the calibration curve of the biosensor cells.

20. The calibration method of a biosensor array according to Claim 19, where the step w) is continuous throughout the steps t) to v).

21. Detection method using a biosensor array according to one or more of Claims 1 to 12,

where the method comprises the following sequential steps y) to D) to define a value of saturation point of each biosensor cells of the biosensor array;

y) pour at least six or more analyte solutions having different concentration and measure drain current changes of the each biosensor cells of the biosensor array

z) plot relations between analyte concentrations and drain current changes

A) calculate 1^st primary approximation line as a curribration curve by using first three or four values of drain current changes

B) calculate 2^nd primary approximation line by using the value of the last three drain current changes,

C) extend both 1^st and 2^nd primary approximation lines to figure out the crossing point of the both lines as a saturation point

D) use the saturation points of each biosensor cells of the biosensor array as the switching point to switch from higher sensitiy biosensor cells to lower sensitivity cells of the biosensor array.