SEMICONDUCTOR BASED BIOSENSOR UTILIZING THE FIELD EFFECT OF A NOVEL COMPLEX COMPRISING A CHARGED NANOPARTICLE

Abstract

The present invention relates to a biosensor for detecting analytes comprising a bio-sensing surface which comprises a field effect transistor and a first binding molecule which is bonded to the surface of the field effect transistor. Furthermore, the biosensor comprises a complex comprising second binding molecules which are conjugated to charged nanoparticles by linker molecules, wherein at least one second binding molecule conjugated to a charged nanoparticle interacts with the first binding molecule wherein the charged nanoparticle is configured to apply a field effect on the field effect transistor. Moreover, the present invention provides a method of detecting an analyte by a biosensor.

Claims

1. A biosensor for detecting analytes comprising a bio-sensing surface which comprises a field effect transistor and a first binding molecule which is bonded to the surface of the field effect transistor; and a complex comprising second binding molecules which bind to the first binding molecule and which are conjugated to charged nanoparticles by linker molecules, wherein, at least one second binding molecule is conjugated to one charged nanoparticle; the at least one second binding molecule conjugated to a charged nanoparticle interacts with the first binding molecule wherein the charged nanoparticle is configured to apply a field effect on the field effect transistor; the affinity of the at least one second binding molecule to the first binding molecule is adaptable such that the first binding molecule releases the complex comprising the at least one second binding molecule in presence of the analyte; and the field effect transistor is configured such that the current measured in dependence of a voltage applied to said field effect transistor is changed due to displacement of the complex comprising the at least one second binding molecule from the first binding molecule by the analyte.

2. A biosensor according to claim 1, wherein the complex comprises a charged nanoparticle selected from a group consisting of metallic nanoparticles, semiconductor nanoparticles, quantum dots or non-metallic nanoparticles, wherein the nanoparticles are charged to carry a positive or negative charge; at least one linker molecule selected from a group consisting of a bond, alkyl, polyethylene glycol (PEG), polyamide, peptide, carbohydrate, oligonucleotide or polynucleotide; and at least one second binding molecule selected from a group consisting of proteins, peptides, nucleic acids or synthetic components.

3. A biosensor according to claim 1, wherein one second binding molecule is conjugated to one charged nanoparticle.

4. A biosensor according to claim 1, wherein the affinity of the at least one second binding molecule to the first binding molecule is less compared to the affinity of the analyte to the first binding molecule.

5. A biosensor according to claim 1, wherein the first binding molecule is selected from proteins, peptides, nucleic acids or antibodies and fragments thereof.

6. A biosensor according to claim 1, wherein the nanoparticle is a metallic nanoparticle and is selected from a group consisting of gold, silver, titanium and platinum, or the nanoparticles are magnetic metallic nanoparticles selected from Fe.sub.3O.sub.4, or wherein the nanoparticle is a semiconductor nanoparticle selected from a group consisting of SiO.sub.2 or the nanoparticle is a quantum dot selected from a group consisting of CdSe/CdS, CdSe/ZnS, InAs/CdSe, ZnO/MgO, CdS/HgS, CdS/CdSe, ZnSe/CdSe, MgO/ZnO, ZnTe/CdSe, CdTe/CdSe and CdS/ZnSe.

7. A biosensor according to claim 6, wherein the nanoparticle is functionalized with SH-PEG-COOH to carry a negative charge; or wherein the nanoparticle is functionalized with SH-PEG-NFh to carry a positive charge.

8. A biosensor according to claim 1, wherein additional charged compounds are conjugated to the charged nanoparticle.

9. A biosensor according to claim 8, wherein charged compounds selected from charged peptides or nucleic acids are conjugated to the charged nanoparticle.

10. A biosensor according to claim 1, wherein Cys-negative charged peptides or Cys-positive charged peptides are conjugated to the charged nanoparticle.

11. A method of detecting an analyte with a biosensor wherein the method comprises the steps of i. providing a biosensor with a bio-sensing surface which comprises a field effect transistor and a first binding molecule which is bonded to the surface of the field effect transistor; ii. selecting a second binding molecule with a lower affinity to the first binding molecule compared to the analyte; iii. conjugating the second binding molecules to charged nanoparticles via linker molecules; iv. bonding the second binding molecules, which are conjugated to charged nanoparticles via linker molecules, to the first binding molecule of the biosensing surface; v. measuring the field effect of the charged nanoparticles to the field effect transistor by measuring the current in dependence of a voltage applied to the field effect transistor; vi. contacting the analyte with the bio-sensing surface and the charged nanoparticles which are conjugated to second binding molecules; vii. measuring the change of the field effect acting on the field effect transistor in presence of the analyte by measuring the current in dependence of a voltage applied to the field effect transistor, wherein the second binding molecules conjugated to charged nanoparticles are partially or completely displaced by analytes due to the higher affinity of the analytes to the first binding molecules, thereby changing the field effect acting on the field effect transistor.

12. The method of detecting an analyte by a biosensor according to claim 11, wherein the concentration of the analyte is calculated by the change of the current in dependence of a voltage applied to the field effect transistor.

13. The method according to claim 11, wherein the second binding molecules and the charged nanoparticles are conjugated by a standard two step procedure.

14. The method according to claim 11, wherein the analyte is present in a physiological solution selected from blood, serum, saliva, urine, stool or plasma.

15. The method according to claim 11, wherein the field effect of the analyte acting on a field effect transistor is lower compared to the field effect of the second binding molecule conjugated to a charged nanoparticle, wherein the field effect of the analyte and of the charged nanoparticle on the field effect transistor is determined by measuring the current in dependence of a voltage applied to the field effect transistor.

16. The biosensor according to claim 1, wherein said biosensor is configured to detect an analyte which is present in a physiological solution selected from blood, serum, saliva, urine stool or plasma.

17. The biosensor according to claim 1, wherein the field effect of the analyte acting on a field effect transistor is lower compared to the field effect of the second binding molecule conjugated to a charged nanoparticle, wherein the field effect of the analyte and of the charged nanoparticle on the field effect transistor is determined by measuring the current in dependence of a voltage applied to the field effect transistor.

Description

[0147] In the following, the present invention is further described by 8 figures and 3 examples.

[0148] FIG. 1 illustrates FET biosensors which are state of the art;

[0149] FIG. 2 (A) illustrates a charged nanoparticle and (B) illustrates the biosensor according to the invention;

[0150] FIG. 3 illustrates the signals measurable with a field effect transistor of a complex according to the invention and an analyte present in a solution with a high salt concentration;

[0151] FIG. 4 (A) illustrates the functionalization of a gold nanoparticle to carry a negative charge and its conjugation with a second binding molecule, (B) illustrates the functionalization of a gold nanoparticle to carry a positive charge and its conjugation with a second binding molecule;

[0152] FIG. 5 (A) illustrates the functionalization of a negative charged gold nanoparticle conjugated to a second binding molecule which is functionalized with additional Cys-negative charged peptides, (B) illustrates the functionalization of a positive charged gold nanoparticle conjugated to a second binding molecule which is functionalized with additional Cys-positive charged peptides;

[0153] FIG. 6 shows the results of the affinity measurements of monoclonal mouse IgG1 anti human CRP antibody B08 against the biomarker CRP (SEQ ID NO: 1) and modified peptide sequences of SEQ ID NOs: 2 to 6;

[0154] FIGS. 1 (A) and (B) illustrate state of the art FET biosensors. FIG. 1 illustrates a semiconductor with an antibody acting as binding molecule on its surface. In FIG. 1 (A) an analyte is bound to the antibody and applies a measurable field effect on the field effect transistor. In several cases, especially when the analyte is present in solutions with high salt concentration, the analyte is bound to the antibody but no measurable field effect is applied to the semiconductor by the analyte, which is due to charge screening effects.

[0155] FIG. 2 (A) illustrates a charged nanoparticle which is conjugated to a second binding molecule. The second binding molecule is bound to a binding molecule on the surface of the field effect transistor, thereby the charged metal particle applies a field effect on the field effect transistor which is measurable (FIG. 2 (B)). If an analyte is added to the biosensor according to the invention the complex comprising the second binding molecule and the charged nanoparticle is displaced by the analyte on the binding site of the first binding molecule on the surface of the field effect transistor. Due to the displacement the field effect applied on the field effect transistor is altered. In case the analyte applies a low or even no field effect on the field effect transistor the measurable field effect on the field effect transistor is decreased. Since the displacement of the complex by the analyte is proportional to the concentration of the analyte, the change of the field effect is a measure for the concentration of the analyte in the solution. Therefore, especially analytes present in solutions with a high salt concentration or physiological solutions like blood, serum, saliva, stool, urine or plasma are detectable.

[0156] FIG. 3 illustrates the signals measurable with a field effect transistor of a complex according to the invention and an analyte present in a solution with a high salt concentration. The figure illustrates the current measured with a constant voltage of a field effect transistor in dependence of the time for two substances S1 and S2. S1 is a biomarker which is present in a high salt solution applying a week field effect on the field effect transistor (dashed line). Substance S2 is a complex according to the invention also present in a high salt solution. As can be seen a significantly higher current is measured with the field effect transistor for S2 (solid line).

[0157] In FIG. 4 (A) a gold nanoparticle is functionalized with a SH-PEG-COOH to carry a negative charge. Subsequently, the negative charged gold nanoparticle is conjugated to a Cys-peptide in a two-step procedure. Firstly, the carboxyl groups of the SH-PEG-COOH functionalized gold nanoparticle are activated by an EDC/NHS activation reaction. Afterwards the NH2-PEG-MAL heterobifunctional reagent is added and a maleimide activated negative charged nanoparticle is obtained. Secondly, a Cys-peptide is added and conjugated to the charged gold nanoparticle.

[0158] FIG. 4 (B) illustrates the procedure for a gold nanoparticle which is functionalized with a SH-PEG-NH.sub.2 to carry a positive charge. Accordingly, the positive charged gold nanoparticle is conjugated to a Cys-peptide in a two-step procedure. Firstly, the NHS-PEG-MAL heterobifunctional reagents is added and a maleimide activated positive charged nanoparticle is obtained. Secondly, a Cys-peptide is added and conjugated to the charged gold nanoparticle.

[0159] Further charged compounds can be added to the complex of the invention. FIG. 5 (A) illustrates the two-step functionalization reaction of negative charged metal nanoparticles. First carboxylated metal nanoparticles are activated by EDC/SulfoNHS reaction and functionalized with an NH.sub.2-PEG-MAL heterobifunctional cross linker. In a second reaction step a Cys-terminated peptide (a second binding molecule) is conjugated in parallel together with a negative charged Cys-peptide (like RRRLC-amid) to the maleimide group. Thereby additional negative charged groups are placed on the metal nanoparticle surface.

[0160] FIG. 5 (B) illustrates the two step functionalization reaction of positive charged metal nanoparticles. First aminated metal nanoparticles are functionalized with a SulfoNHS-PEG-MAL heterobifunctional cross linker. In a second reaction step a Cys-terminated peptide (a second binding molecule) is conjugated in parallel together with a positive charged Cys-peptide (like CLDDD-OH) to the maleimide group. Thereby additional positive charged groups are placed on the metal nanoparticle surface.

EXAMPLES OF THE INVENTION

Example 1—Functionalization of Gold Nanoparticles

[0161] The functionalization is performed by the gold (metal)-thiol reaction using either SH-PEG-COOH heterobifunctional reagents with a molecular weight of 400 Da. A 10 mg/ml SH-PEG-COOH (MW 634.77 g/mol) is added to 10 nM of gold nanoparticles having a diameter of 15 nm (functionalization works in the same way also for gold nanoparticles having a diameter of 20 nm, 10 nm or 5 nm) and incubated for 4-24 hours at RT. After the metal-thiol reaction is completed the Au nanoparticles are washed in water and PBS. The stability of the Au particles is determined by an UV/VIS spectral analysis. Stable Au nanoparticles show a high absorption at 520 nm and no absorption at 700 nm, whereas instable Au nanoparticles show a great absorption at 700 nm and a decreased absorption at 520 nm.

Example 2—Two-Step Procedure to Conjugate a Second Binding Molecule and a Negative Charged Gold Nanoparticle

[0162] A gold nanoparticle is functionalized with SH-PEG-COOH to carry a negative charge. The charged gold nanoparticle shall be conjugated to a second binding molecule (which is a peptide) via a thiol-maleimide reaction in a two-step procedure according to the invention.

[0163] Step 1

[0164] The functionalization is performed by a Mal-PEG-NH2 heterobifunctional reagents. In order to react the amino (NH2) group of the heterobifunctional cross linker with the carboxyl groups of the nanoparticle, an EDC/Sulfo NHS activation reaction is required. Therefore 0.4 mg EDC and 1.1 mg Sulfo-NHS are added to 100 μl of 10 nM gold nanoparticles for 15 min at room temperature. After activation of carboxyl groups the reaction mixture is purified from excess reagent by molecular weight cut-off columns and transferred into PBS pH 7.2. Immediately after purification a 10-100 fold molar excesses of Mal-PEG-NH2 heterobifunctional cross linker is added for 30 min at room temperature. The excess reagents are purified off by molecular weight cut-off columns.

[0165] Step 2

[0166] The purified nanoparticle-PEG-Mal conjugate is directly used for the peptide conjugation reaction. The reaction takes place at a pH between 6.5 and 7.5. Thiol-containing compounds, such as dithiothreitol (DTT) and beta-mercaptoethanol (BME), are excluded from reaction buffers used with maleimides because they will compete for coupling sites.

[0167] DTT, which is used to reduce disulfides, to make sulfhydryl groups available for conjugation is thoroughly removed using a desalting column before initiating the maleimide reaction. Since the disulfide-reducing agent TCEP does not contain thiols it is not removed before reactions involving maleimide reagents. Excess maleimides are quenched at the end of a reaction by adding free thiols. EDTA is included in the coupling buffer to chelate stray divalent metals that otherwise promote oxidation of sulfhydryls (non-reactive). The conjugated peptides are added in a 10-1,000 fold molar excess. The conjugation reaction takes place at room temperature for 2-4 hours. The excess reagents are purified off by molecular weight cut-off columns.

Example 3—Two-Step Procedure to Conjugate a Second Binding Molecule in Parallel Together with Additional Negative Charged Molecules to a Negative Charged Gold Nanoparticle

[0168] A gold nanoparticle is functionalized with SH-PEG-COOH to carry a negative charge. The charged gold nanoparticle shall be conjugated to a second binding molecule (which is a peptide) and to an additional negative charged molecule (which is a peptide of the sequence: RRRLC-OH) via a thiol-maleimide reaction in a two-step procedure according to the invention.

[0169] Step 1

[0170] The functionalization is performed by a Mal-PEG-NH2 heterobifunctional reagents. In order to react the amino (NH2) group of the heterobifunctional cross linker with the carboxyl groups of the nanoparticle, an EDC/Sulfo NHS activation reaction is required. Therefore 0.4 mg EDC and 1.1 mg Sulfo-NHS are added to 100 μl of 10 nM gold nanoparticles for 15 min at room temperature. After activation of carboxyl groups the reaction mixture is purified from excess reagent by molecular weight cut-off columns and transferred into PBS pH 7.2. Immediately after purification a 10-1,000 fold molar excesses of Mal-PEG-NH2 heterobifunctional cross linker is added for 30 min at room temperature. The excess reagents are purified off by molecular weight cut-off columns.

[0171] Step 2

[0172] The purified nanoparticle-PEG-Mal conjugate is directly used for the peptide conjugation reaction. The reaction takes place at a pH between 6.5 and 7.5. Thiol-containing compounds, such as dithiothreitol (DTT) and beta-mercaptoethanol (BME), are excluded from reaction buffers used with maleimides because they will compete for coupling sites.

[0173] The conjugated peptides are added in a 10-1,000 fold molar excess whereby the additional negative charged molecule has a 5-20 fold molar excess compared to the second binding molecule. This means if the second binding molecule is used in 10 fold molar excess, the additional negative charged molecule has a 50-200 fold molar excess compared to the gold nanoparticle concentration. The conjugation reaction takes place at room temperature for 2-4 hours. The excess reagents are purified off by molecular weight cut-off columns.

Example 4—Coupling of a First Binding Molecule on swCNTs

[0174] The single walled CNT (swCNT) network is present on a biosensor surface and shall be functionalized with a first binding molecule (an antibody).

[0175] First a 1 mM 1-pyrenebutric acid solution in EtOH is incubated for 1-24 hours at room temperature. The excess reagent is purified off by washing the sensor 3 times with EtOH followed by a subsequent 3 times washing step with water. The functionalization of the antibody is performed by coupling the antibody amino groups with the carboxyl group of the 1-pyrenebutric acid. Consequently the carboxyl groups of the 1-pyrenebutric acid have to be activated by an EDC/Sulfo NHS activation reaction. Therefore 0.4 mg EDC and 1.1 mg Sulfo-NHS are added to 1 ml of an amino free buffer like PBS (pH 6.0). The activation reaction takes place for 15 minutes at room temperature.

[0176] Directly after the activation reaction the antibody is added in a concentration of 1-0.1 mg/ml to the biosensor at pH 7.2-8.0 for 1-4 hours at room temperature. The excess antibody is purified off by washing the sensor 3 times with PBS pH 7.2.

Example 5—Test of Sensor Functionality

[0177] The current sensor chips are conducted by crocodile clamps to a dual-channel source meter (Keithley 2612B). The samples are applied by a pipet. Sample volumes are varied between 10 and 50 μl.

[0178] As gate electrode a Ag/AgCl electrode operating in a top gate setting was used. A feedback circuit was also implemented, which measures constantly the applied gate current and regulates the gate current voltage if necessary.

[0179] In a pre-test the sensitivity to fluids with different pH-values was tested. As result it was found that the sensor reacts very strongly and reliably to a change between pH 6 and pH 7 solutions.

[0180] To test the measurement set up and the general sensor functionality different pH PBS buffers were subsequently applied on the sensor surface. Therefore, a PBS solution (pH 7) was mixed with 20 nm Au nanoparticles (Au-NP) and a concentration of 2.4 pM. The same PBS solution without Au-NP served as reference. It could be shown that there is a significant sensor response when the two fluids are exchanged cyclically. This confirms that the semiconductor sensor is influenced by low concentrations of Au-NP.

Example 6—Measurement of the Biomarker C-Reactive Protein (CRP)

[0181] A complex comprising a second binding molecule that is coupled to Au nanoparticle via a linker has been produced as described in example 1. 5 nM Au particles with 10 functionally coupled peptides per Au particle were used in the experiment.

[0182] The biosensor was functionalized as described in example 4, in this experiment with the monoclonal mouse IgG1 anti human CRP antibody B08. The affinity of the antibody was first tested against CRP (SEQ ID NO: 1) and the modified peptide sequences of SEQ ID NOs: 2 to 6. Sequences are shown in the following table:

TABLE-US-00001 Amino acid Peptide-ID sequence SEQ ID NO: Original sequence of CVFPKESD 1 CRP 80712 CAFPKESD 2 80713 CVFPRESD 3 80714 CVFPKDSD 4 80715 CVFPKETD 5 80716 CVYPKESD 6

[0183] The results of the affinity measurements are shown in FIG. 6. The subsequent displacement measurements were performed with the peptide 80715 (SEQ ID NO: 5).

[0184] The Au nanoparticles are bound to the CRP-specific antibody via a peptide and exert a field effect on the semiconductor. If the biomarker (in this case CRP) is present in the blood sample, it can displace the nanoparticle and thus annul the field effect.

[0185] Results of the measurement of a displacement reaction: The current/voltage curve shows the change in the transistor property (by annulling the field effect). First PBS without biomarker was added, then the concentration of the biomarker CRP in PBS was gradually increased. By adding different concentrations of the biomarker CRP, the current-voltage curve of the transistor has changed accordingly. The following CRP concentration s were used: 381 fM, 3 pM, 24 pM, 195 pM, 1.56 nM, 12.5 nM, 100 nM and 800 nM. The voltage changes measured are shown in the following table:

TABLE-US-00002 CRP concentration ΔV (V) 381 fM −0.009 3 pM −0.013 24 pM −0.016 195 pM −0.017 1.56 nM −0.020 12.5 nM −0.022 100 nM −0.024 800 nM −0.027

[0186] At constant current, changes in the biomarker concentration were measured as voltage changes. It was found that there is a nearly linear relationship between voltage change (ΔV in V) and CRP concentration. The biomarker CRP could be reliably detected in a concentration range between 800 nM to 381 fM. Reaction times were 10 minutes, measurements were performed in PBS (i.e. 150 mM salt concentration).

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