Electrochemical aptasensors with a gelatin B matrix

Abstract

This invention provides: —an aptamer-based electrochemical sensor, wherein said aptamer is covalently bonded to or chemisorbed on an electrode, said aptamer forming a complex with a target molecule and is encapsulated by a gelatin B matrix; —a method of manufacturing said aptamer-based electrochemical sensor; —the use of the aptamer-based electrochemical sensor for the electrochemical determination of a concentration of a target molecule; and —a composite electrode combining a polymeric material and electrically conducting particles for selective analyte detection, wherein said electrode is coated with gelatin type B.

Claims

1. An aptamer-based electrochemical sensor for detecting a target molecule, comprising an electrode, wherein an aptamer is covalently bonded to or chemisorbed on said electrode, said aptamer forming a complex with said target molecule, characterized in that said complex is encapsulated by a type B gelatin matrix.

2. An aptamer-based electrochemical sensor according to claim 1, wherein the electrode comprises a gold electrode or a composite electrode combining a polymeric material and electrically conducting particles.

3. An aptamer-based electrochemical sensor according to claim 1, wherein the electrode is coated with gelatin type B.

4. An aptamer-based electrochemical sensor according to claim 1, wherein said target molecule is selected from the group consisting of interferon γ, cell growth factors, antigens, therapeutic drugs, diagnostic agents, antibiotics, toxins, vitamins, recreational drugs, catecholamines, metabolites, proteins and cells.

5. An aptamer-based electrochemical sensor according to claim 1, wherein the aptamer is a DNA aptamer.

6. An aptamer-based electrochemical sensor according to claim 1, wherein said aptamer is 5′-SH-(CH.sub.2).sub.6-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-3′ (SEQ ID NO: 4) or 5′-GTC-TCT-GTG-TGC-GCC-AGA-GAA-CAC-TGG-GGC-AGA-TAT-GGG-CCA-GCA-CAG-AAT-GAG-GCC-C-spacer-NH.sub.2-3′ (SEQ ID NO: 3).

7. A method of manufacturing an aptamer-based electrochemical sensor for determining a concentration of a target molecule comprising the steps of: selecting an aptamer to form a complex with a target molecule; synthesizing said aptamer; adsorbing said aptamer on, or covalently coupling said aptamer with, an electrode; and further providing a gelatin B matrix for said aptamer on said electrode.

8. A method according to claim 7, wherein said target molecule is selected from the group consisting of interferon γ, cell growth factors, antigens, therapeutic drugs, diagnostic agents, antibiotics, toxins, vitamins, recreational drugs, catecholamines, metabolites, proteins and cells.

9. A method for the electrochemical determination of a concentration of a target molecule, comprising the steps of: selecting an aptamer to form a complex with the target molecule; providing an electrode and a gelatin B matrix for said aptamer on said electrode, and adsorbing said aptamer on, or covalently coupling said aptamer to, said electrode.

10. A method according to claim 9, wherein said electrochemical determination is a potentiometric determination or an amperometric determination.

11. A method according to claim 9 for the electrochemical determination of a concentration of chloramphenicol.

12. A method according to claim 9 for the electrochemical determination of a concentration of cocaine.

13. A method according to claim 9 for the electrochemical determination of a concentration of dopamine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a representation of a gelatin-containing aptasensor for CAP detection on a gold (Au) electrode.

(2) FIG. 2 shows cyclic voltammograms of Au (1), Gel B|APT|Au electrode (2) in blank solution and Au (3), APT|Au (4) and Gel B|APT|Au (5) electrode in 1×10.sup.−6 mol L.sup.−1 of CAP solution.

(3) FIG. 3 shows the electrochemical impedance spectra of Au (1), APT|Au (2) and Gel B|APT|Au (3) electrode in 10×10.sup.−3 mol L.sup.−1 [Fe(CN).sub.6].sup.4−/3− in the frequency range 0.1 to 100,000 Hz.

(4) FIG. 4 shows SEM images of the working surface area of a gold electrode after gelatin B immobilization (a), and after aptamer incorporation (b).

(5) FIG. 5 shows in (A) the dependence of the redox peak current on the concentration of CAP at a Gel B|APT|Au electrode, and in (B) calibration curves obtained at APT|Au (1) and Gel B|APT|Au (2) electrodes.

(6) FIG. 6 shows in confocal images of hydrogels. (A): pure Gelatin B; (B): Gelatin B+coupling agents (EDC and NHS); and (C): Gelatin B+coupling agents+fluorescent aptamer. Confocal features: pixels 7.92; red value 5.58; the scale bar (white)=100 μm.

(7) FIG. 7 shows sensorgram recordings for DA on a biosensor with a Gelatin B membrane with covalently linked aptamer. Square concentration pulses were injected for 80 s. The concentration varied from 5×10.sup.−7 M (lower curve) to 10.sup.−6 M, 5×10.sup.−6 M, 10.sup.−5 M, 5×10.sup.−5 M, and 10.sup.−4M (upper curve).

(8) FIG. 8 shows normalisation of the curves in FIG. 7 from time 0 to the time corresponding to maximum response R.sub.max to a 10.sup.−4 M DA injection, to show the variation with concentration.

(9) FIG. 9 shows a Nicolskii-Eisenmann-type calibration graph. The smooth curve is obtained from a non-linear least-squares fit to a Nicolskii-Eisenmann function of the type E=E°+S. log (c.sub.DA+Cst). E°=387 mV, S=60.4 mV and Cst=3.9×10.sup.−7 M.

(10) FIG. 10 shows the differences between the aptamer-based potentiometric biosensor (lighter upper trend line) and the negative control (darker lower trend line, only containing a Gelatin B membrane) in respect of the potentiometric responses of 5×10.sup.−5M to 10.sup.−7 M DA injections in FIA after transformation to a concentration-related signal.

(11) FIG. 11 shows the differences between the aptamer based potentiometric biosensor (lighter), and the negative control (darker) for injections of DA 10.sup.−6 M on a sensor which contains an aptamer (upper curve: lighter) and on a sensor which does not contain an aptamer (lower curve: darker).

(12) FIG. 12 shows the slope of

(13) $\frac{d\left(\mathrm{tR}\right)}{dt}$
versus tR (Transformed Response) curves (see equation (4) below) plotted against the DA concentrations which were used to record the sensorgrams.

(14) FIG. 13 shows the manufacture of a gelatin B-containing aptasensor based on a gold screen printed electrode (SPE) for the detection of CAP by means of differential pulse voltammetry.

(15) FIG. 14 shows electrochemical impedance spectra obtained with the bare SPE of FIG. 13 (FIG. 14a), the aptamer modified SPE (FIG. 14b) and the aptamer/gelatin B modified SPE (FIG. 14c).

(16) FIG. 15 shows the differential pulse voltammograms of accumulated CAP (10.sup.−9 M) at the surface of the bare gold SPE of FIG. 13 (curve a), a gelatin A modified SPE (curve b), a gelatin B modified SPE (curve c), an aptamer modified SPE (curve d), an aptamer/gelatin A modified SPE (curve e) and an aptamer/gelatin B modified SPE (curve 0 in tris buffer solution.

(17) FIG. 16 shows the influence of the kind (FIG. 16 A) and time (FIG. 16 B) of accumulation of CAP on the electrochemical detection signal.

(18) FIG. 17 shows in (A) the dependence of the redox peak current on the concentration of CAP at a Gel B|APT|Au SPE electrode, and in (B) calibration curves obtained at APT|Au SPE (1) and Gel B|APT|Au SPE (2) electrodes.

ELECTRODE

(19) The electrochemical aptasensor according to the present invention requires at least a detecting electrode, also named a working electrode. A conventional electrochemical device is a three-electrode cell configuration comprising a reference electrode (such as, but not limited to, a calomel electrode or a silver electrode) and a counter-electrode. Suitable detecting electrodes include gold electrodes, glassy carbon electrodes, an inert metal in an ionically conducting composite, and composite electrodes combining a polymeric material and electrically conducting particles. The electrode may be obtained by any manufacturing process known in the art, including the screen printing technique for making a SPE.

(20) WO 2005/103664, the content of which is incorporated herein by reference, discloses suitable composite potentiometric electrodes for selective analyte detection—according to the present invention, provided that said electrodes are coated with an ionically conductive hydrophilic, preferably negatively charged, matrix such as gelatin type B or an equivalent thereof, for instance in the form of a thin (μm scale) or ultrathin (nm scale) layer. Gelatin B for performing this aspect of the invention may have any Bloom number, including the low range 50-125, the medium range 125-225 and the high range 225-325.

(21) This composite electrode, and a potentiometric cell including it, may be for an in vivo analyte sensor or an in vitro analyte sensor.

Aptamer-Based Electrochemical Sensor

(22) In a first aspect of the present invention, the above objective is realised by an aptamer-based electrochemical sensor, wherein said aptamer is covalently bonded to or chemisorbed on an electrode, said aptamer is selected to form a complex, usually a robust complex with a target molecule and is encapsulated by an ionically conductive hydrophilic, preferably negatively charged, matrix such as gelatin B or an equivalent thereof. Gelatin B for performing this aspect of the invention may have any Bloom number, including the low range 50-125, the medium range 125-225 and the high range 225-325.

(23) According to a preferred embodiment of the first aspect of the present invention, said aptamer is selected with the SELEX procedure, or it may be known from the literature.

(24) According to another preferred embodiment of the first aspect of the present invention, said target molecule is selected from the group consisting of vitamins, antibiotics, toxins, therapeutic drugs, diagnostic agents, recreational drugs (e.g. cocaine), catecholamines, metabolites, proteins and cells.

(25) According to another preferred embodiment of the first aspect of the present invention, said aptamer is 5′-SH-(CH2)6-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-3′ (SEQ ID NO: 4) and is intended for CAP detection.

(26) According to another preferred embodiment of the first aspect of the present invention, said aptamer is 5′-GTC-TCT-GTG-TGC-GCC-AGA-GAA-CAC-TGG-GGC-AGA-T A T-GGG-CCA-GCA-CAG-AA T-GAG-GCC-C-spacer-NH2-3′ (SEQ ID NO: 3) and is intended for dopamine detection.

Method of Manufacturing an Electrochemical Sensor

(27) In a second aspect of the present invention, the above objective is realised by a method of manufacturing an aptamer-based electrochemical sensor for determining a concentration of a target molecule comprising the steps of: selecting an aptamer to form a complex, preferably a robust complex, with a target molecule, e.g. using the SELEX procedure; synthesizing said aptamer; adsorbing said aptamer on or covalently coupling said aptamer with an electrode; and providing an ionically conductive hydrophilic, preferably negatively charged, matrix, preferably gelatin type B or an equivalent thereof, for said aptamer on said electrode.

(28) According to a preferred embodiment of the second aspect of the present invention, said target molecule is selected from the group consisting of vitamins, antibiotics, toxins, therapeutic drugs, diagnostic agents, recreational drugs, catecholamines, metabolites, proteins and cells.

Use of the Aptamer-Based Electrochemical Sensor

(29) In a third aspect of the present invention, the above objective is realised by the use of the aptamer-based electrochemical sensor according to the first aspect of the present invention or produced according to the second aspect of the present invention for the electrochemical determination of a concentration of a target molecule.

(30) According to a preferred embodiment of the third aspect of the present invention, said electrochemical determination is a potentiometric determination.

(31) According to another preferred embodiment of the third aspect of the present invention, said electrochemical determination is an amperometric determination.

EXAMPLES

(32) Chemicals and Materials

(33) Promazine, lidocaine, ritodrine and chloramphenicol (CAP) were obtained from Sigma-Aldrich (Bornem, Belgium) and dopamine (DA) was obtained from Fluka. To dissolve these drugs, small amounts of ethanol (Fluka, analytical grade) were used.

(34) MES was obtained from Acros Organics (Eupen, Belgium).

(35) The coupling agents EDC and NHS were obtained from Sigma-Aldrich.

(36) All chemicals were of analytical reagent grade.

(37) Type B gelatin (Gel, IEP=5, Bloom strength=257), isolated from bovine skin by the alkaline process, was provided by Tessenderlo Chemie (Belgium).

(38) Buffers:

(39) Tris buffer containing:

(40) NaCl 100×10.sup.−3 mol L.sup.−1

(41) Tris HCl 20×10.sup.−3 mol L.sup.−1

(42) MgCl.sub.2 2×10.sup.−3 mol L.sup.−1

(43) KCl 5×10.sup.−3 mol L.sup.−1

(44) CaCl.sub.2 1×10.sup.−3 mol L.sup.−1

(45) with a pH of 7.6 obtained from VWR (Belgium) and used as a binding buffer solution.

(46) Aptamers:

(47) binding aptamer sequence (5′-SH-(CH2)6-AGC-AGC-ACA-GAG-GTC-AGA-TGA-CTG-AGG-GCA-CGG-ACA-GGA-GGG-CAT-GGA-GAG-ATG-GCG-3′) (SEQ ID NO: 4) from Eurogentec.

(48) A 58 mer aptamer selected specifically to detect DA (Zheng et al., 2011) (5′-GTC-TCT-GTG-TGC-GCC-AGA-GAA-CAC-TGG-GGC-AGA-TA T-GGG-CCA-GCA-CAG-AAT-GAG-GCC-C-spacer-NH2-3′) (SEQ ID NO: 3) aptamer synthesized by Integrated DNA Technologies (Leuven, Belgium)

(49) Voltammetric Measurements

(50) Voltammetric measurements were recorded by a μ-AutolabII potentiostat controlled by NOVA 1.7 software package (Metrohm, The Netherlands). Electrochemical impedance spectroscopy (EIS) measurements were performed by using a frequency response analyzer module. A gold electrode inlaid disk (Φ=1.6 mm) was used as working electrode. A saturated calomel electrode (SCE) and graphite were used as the reference and the auxiliary electrode, respectively.
Morphological Investigation

(51) Morphological investigation of the electrode surface was performed using a JEOL JSM-6300 Scanning Electron Microscope (SEM) and confocal microscopy was performed by localizing Cy3 dyes with a Nikon C1 laser scanning confocal unit (D-eclipse-C1, Nikon, Melville, N.Y.) equipped with an argon and a helium/neon laser line fitted onto an upright microscope (Eclipse E600, Nikon, Melville, N.Y.) in combination with a 10× planfluor (NA: 0.50) objective manufactured by Nikon (Melville, N.Y.).

(52) FIA

(53) The FIA recordings were performed using a LC-10ADvp pump (Shimadzu Liquid Chromatography) and a Rheodyne 7125 six port external sample injector (VICI, US). A 1.00 mL sample loop was used to generate square concentration pulses for sensorgram recording in FIA conditions. The PEEK tubing (Alltech, USA) of the injection loop and the injector-detector connections had an internal diameter of 0.18 mm. The flow rate was 1.00 mL min.sup.−1. Poiseuille peak broadening effects were kept to a minimum using short injector-detector connections (150 mm). To avoid such effects at the end of the square concentration pulse, the injector was switched from inject to load after 80 s (well before the sample loop volume was totally emptied).

(54) This results in a sharp pulse with negligible broadening as well at the start as at the end of the pulse. The eluent was 10.0 mM MES, pH 7.0.

(55) The column outlet was directed perpendicularly towards the sensitive membrane of the coated wire electrode in a “wall-jet” flow cell. The distance from the LC tubing outlet to the electrode was 0.10 mm.

(56) Preparation of the Amperometric Electrode for Example 1

(57) Prior to surface modification, the gold electrode was mechanically polished with 1.0 and 0.05 μm alumina slurry separately, followed by rinsing thoroughly with double distilled water. Then it was washed ultrasonically in double distilled water then ethanol for 15 minutes. The electrode was rinsed with distilled water and dried at room temperature. Subsequently, electrodes were electrochemically cleaned by potential scanning between −1.2 and 1.2 V until a reproducible cyclic voltammetric scan was obtained.

(58) To immobilize the CAP-binding thiolated aptamer on the gold surface, 3×10.sup.−6 L of a 2.5×10.sup.−6 mol L.sup.−1 aptamer and 5% (w/w) gelatin type B solution was dropped onto a freshly smoothed gold surface, and the solvent was then evaporated at 4° C. for 6 hours. The final sensing interface was ready after rinsing with buffer solution.

(59) Preparation of the Potentiometric Electrode for Example 2

(60) The indicator electrode is made of a PVC cylinder. It contains a cylindrical substrate electrode (3.0 mm diameter×1.0 mm length), which is an electronically conducting graphite/PVC composite material. The composite substrate electrode was polished with Carbimet grid 600 (Buehler Ltd, USA).

(61) To coat the gelatinous hydrogel on the electrode, 10.0 μL of a mixture, which consists of 25.0 mg gelatin B dissolved in 0.50 mL 10.0 mM MES (5% w/v) pH 7.0 at 40° C., was brought onto the electrode surface with a micropipette and exposed to air for 2 hours at 4° C. (drop drying).

(62) The EDC-NHS coupling procedure was used to bind the DA-binding aminated aptamer covalently to the gelatin B hydrogel (which contains carboxyl groups). After adding 20 μL of the coupling agents (15.32 mg of EDC and 2.32 mg of NHS dissolved in 100 μl of 10.0 mM MES buffer at pH 7.0) to the coated hydrogel for 2 hours, 2×4 μL of the aptamer (10.sup.−4M) was applied for 1 hour. After evaporation for 2 hours at 4° C. the electrodes were kept in 10.0 mM MES running buffer, pH 7.0 for at least 3 hours until a stable baseline was obtained. The sensorgrams of each analyte were measured on 3 electrodes, and used when the inter-electrode reproducibility was better than 10%. At least 3 sensorgrams (injections) were recorded on each electrode after conditioning and stabilization in the running buffer.

(63) The membrane potential was measured against an Orion 800500Ross® reference electrode (Ag/AgCl) using a high impedance (10.sup.13Ω) homemade amplifier. The detection signals were recorded on a data station composed of a computer equipped with a 6013 NI DA converter and LabVIEW 7 (National Instruments, US) based software. The overall RC time constant of the high impedance amplifier plus data station was set to 0.2 s.

Example 1—Electrochemical CAP-Aptasensor

(64) Morphology

(65) The morphology of the prepared electrodes was investigated by SEM measurements (see FIG. 4). The SEM image of a gelatin assembled gold electrode is shown in FIG. 4(a), a regular and porous structure being observed. The gelatin film was compact, smooth and homogenous without grainy and porous structure, showing that an ordered matrix was formed. However, a rough, sponge-like and irregular surface appeared when aptamers were added to the matrix, indicating a clear morphology change due to the interactions.

(66) Electrochemical Behaviour:

(67) Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were performed to characterize an APT|Au electrode and a Gel B|APT|Au electrode as shown in FIGS. 2 and 3 respectively.

(68) Whereas at a bare gold electrode no obvious redox peak was observed in the CV-characteristic (indicated by 3 in FIG. 2), a large reduction peak was observed in the CV-characteristic (indicated by 4 in FIG. 2) at APT|Au. With gelatin type B encapsulation the response (indicated by 5 in FIG. 2) increased due to the increased electron transfer capability of the modified electrode. A gelatin type B film acts as both a supporting polymer for aptamers and as a reaction medium. Therefore, substrate mass transport occurred in and out of the hydrogel layer while the aptamers were retained in the matrix and on the electrode surface.

(69) The EIS characteristics shown in FIG. 3 demonstrate that the electron transfer resistance increased in the following order: Au electrode (indicated by 1 in FIG. 3), APT|Au electrode (indicated by 2 in FIG. 3) and Gel B|APT|Au electrode (indicated by 3 in FIG. 3). This is attributed to the fact that ssDNA with negative charges on its phosphate backbone makes an electrostatic repulsive force to the [Fe—(CN).sub.6].sup.3−/4− anions and prevents electrons from reaching the electrode surface. The increase in electron transfer resistance indicates that the aptamers are successfully immobilized on the electrode surface. In the case of a Gel B|APT|Au electrode, an additional barrier of negatively charged gelatin has increased the electron transfer resistance which results in a larger semi-circle. This demonstrates the successful immobilization of APT on the electrode and the blocking effect of the gelatin layer to unspecific redox active molecules. The CV-characteristics of the resulting aptasensor at different scan rates were also studied. In the presence of CAP molecules, a linear dependence of peak current upon scan rate implied that the electrochemical process was a surface confined process. The short-term stability of the aptasensor was investigated over successive cyclic voltammograms. The response current of the electrode with immobilized aptamer and gelatin encapsulation decreased by about 3.2% of its initial response and the relative standard deviation (R.S.D.) was 1.8%, whereas the electrode only with immobilized aptamer showed a 20.2% decrease and an R.S.D. of 3.5%. The high stability could be attributed to the high chemical stability of gelatin and aptamers, both improving the stability of the aptasensor. The good stability of the Gel B|APT|Au can be ascribed to excellent biocompatibility and the stabilizing microenvironment around the aptamers provided by the modified gelatin sensors.

(70) From FIG. 5 (A), it can be seen that the peak current increases with increasing CAP concentration. There was a linear relationship between peak current and CAP concentration in the range from 2.1×10.sup.−9 to 5.2×10.sup.−7 mol L.sup.−1. The linear regression equation was I=−1.6 C+2.7, (units for C and I are ×10.sup.−9 M and ×10.sup.−7 A respectively) and the correlation coefficient is 0.9684. The detection limit was 1.2×10.sup.−9 mol L.sup.−1 based on interpolation to the point where the current differs from the background current for three standard deviations calculated from the currents obtained for three different electrodes. In the case of APT|Au electrodes (without gelatin as a protective matrix), the detection limit was found to be 1.6×10.sup.−9 and the linear range was between 1.6×10.sup.−9 and 4.2×10.sup.−7 mol L.sup.−1 which demonstrated the improved performances of the Gel B|APT|Au electrode over the APT|Au electrode. The enhanced sensitivity and broader dynamic range could be due to the ability of gelatin B to make diffusion of analytes easier and prevent aptamers from leaking and most important, prevent the electrode surface from undesired adsorption. A comparison of the calibration curves for the two electrodes [see FIG. 5(B)] shows that the sensitivity (slope) of the Gel B|APT|Au electrode was higher than that of the APT|Au electrode.

(71) The assay of this target in real samples was investigated by detecting CAP in a sample of skimmed cow's milk. The standard addition method was employed to evaluate the applicability of the developed aptasensor. The increased reduction peak of CAP compared with the reduction current obtained at an aptamer immobilized electrode without gelatin as protective matrix occurred at a Gel B|APT|Au electrode in the expected potential range, which indicated an enhanced sensitivity of the developed Gel B|APT|Au electrode. Recovery values between 87% and 94% were obtained, indicating the applicability of the developed aptasensor for CAP detection in real biological samples.

Example 2—Electrochemical DA-Aptasensor

(72) Morphology

(73) The covalent binding of the aminated oligonucleotides to the gelatin B, which contains carboxyl groups, was examined by confocal microscopy. To exclude the background signal, pure Gelatin B and Gelatin B treated with coupling agents (EDC and NHS), were checked as blanks. No signal was observed in the latter cases (FIGS. 6A and 6B). This is in contrast with the bright fluorescent signal observed for a hydrogel treated with a fluorescent labeled (Cy3) aminated aptamer (FIG. 6C). Even after three hours of use in the FIA potentiometric set-up, the intense fluorescence persisted. This experiment confirmed the covalent coupling of the aminated oligonucleotides to the gelatin B.

(74) Electrochemical Behaviour

(75) After checking the coupling of the aptamer to the biopolymer as described above, the electrodes were placed in a FIA potentiometric setup. After injection of an analyte in the FIA system, the voltage output varies with time, due to the kinetics of complex formation between DA and the anti-DA aptamer. A positively charged analyte molecule such as DA has the tendency to adsorb to the sensor coating, if a DA binding aptamer is present in this material. This provokes a surface potential change, which is our analytical signal. In its most simple representation, the eluent/gelatin B interface is expected to have a Boltzmann type distribution of positive charges as the gelatin B behaves as a cation exchange-like material in the given conditions.

(76) The target molecules were injected as square concentration pulses, comparable with the block pulses in SPR. The sensorgrams (mV responses as a function of time) obtained are shown in FIG. 7. Using a hydrodynamic method and square concentration pulses has the advantage that both adsorption as desorption curves can be obtained and the adsorption/desorption kinetics calculated.

(77) FIG. 7 shows the potentiometric response of DA on the sensor which contains the DA specific aptamer. The response heights (mV obtained at the plateau values after 80 s of injection) are concentration dependent: see FIGS. 7 and 9. After normalization of the curves from time zero to R.sub.max (see FIG. 8), it is clear that higher concentrations of DA show faster “on” kinetics than lower concentrations. This concentration dependent difference in adsorption kinetics (rising part of the curve) is typical for SPR experiments.

(78) If the maximum responses (in mV) are plotted against the logarithm of the concentration, the typical Nicolskii-Eisenmann curve is obtained (see FIG. 9). At higher concentration values (results not shown), saturation of the signal (reaching a plateau value) starts to occur. This is ascribed to the fact that a relatively low aptamer concentration (10.sup.−4M) was used to bind to the hydrogel. DA concentrations below 5×10.sup.−5M were therefore used in these experiments.

(79) The Nicolskii-Eisenmann equation (see FIG. 9) was transformed to equation 1, to obtain a concentration dependent output signal (“Transformed Response”, tR) of the sensor.
tR=(10.sup.mV/S−1).Math.Cst  (1)

(80) The transformed R.sub.max values (equation 1) of the potentiometric sensors with coupled aptamer were compared with the values obtained with electrodes which did not have an aptamer coupled to the hydrogel: see FIG. 9. Four electrodes were tested, each with 3 injections. From five to ten times higher signals were measured with the aptamer-containing electrodes. Not only the height, but also the shape of the sensorgram was different when comparing gelatin B membranes with and without coupled aptamer (see FIG. 9). The gelatin B electrodes containing aptamer always yielded slowly falling curves. This is additional evidence for the recognition by the aptamer of the target molecule. Slowly falling curves mean slow desorption kinetics. This yields small k.sub.off values, resulting in high K.sub.association values: see equation. 2.

(81) $\begin{array}{cc}{K}_{\mathrm{association}}=\frac{k_{\mathrm{on}}}{k_{\mathrm{off}}}& \left(2\right)\end{array}$

(82) FIGS. 9, 10 and 11 clearly show that the aptamer-based sensors are very sensitive, the responses being are at least a factor of 800 higher (on a mV basis) than those of the classical PVC-based electrodes.

(83) Determination of the Association Constant, K.sub.ass, Between DA-Specific Aptamer and DA

(84) The above-defined “sensorgram methodology” was used. Sensorgrams were recorded at different analyte concentrations. The rising parts of the sensorgrams are shown in mV in FIG. 8. The mV y-axis in these sensorgrams was first converted to a concentration dependent response, called transformed Response (see equation. 1). The transformed Response (tR) of the sensor was linearly related to the number of occupied adsorption sites, R.sub.occupied, i.e.: transformed Response (tR)˜R.sub.occupied.

(85) The rate of adsorption of DA, v.sub.on, can be regarded as a reaction rate which is first order in the DA concentration in the bulk of the solution (c.sub.analyte) and in the concentration of free adsorption sites (or aptamers) on the sensor surface: R.sub.max−R.sub.occupied. It can be described by a rate equation of the form of equation 3:

(86) $\begin{array}{cc}{v}_{\mathrm{on}}=\frac{d{R}_{\mathrm{occupied}}}{dt}=k_{\mathrm{on}}.Math.c_{\mathrm{analyte}}\left(R_{\max }-R_{\mathrm{occupied}}\right)-k_{\mathrm{off}}{R}_{\mathrm{occupied}}& \left(3\right)\end{array}$

(87) This equation can be rewritten by substituting the concentration of DA molecules, which adsorbed onto (or “occupied”) the aptamer derivatized surface, R.sub.occupied, by the sensor's transformed Response, tR, and by replacing R.sub.max by tR.sub.max. This yields equation 4:

(88) $\begin{array}{cc}\frac{d\left(\mathrm{tR}\right)}{dt}=k_{\mathrm{on}}.Math.c_{\mathrm{analyte}}.Math.{d\left(\mathrm{tR}\right)}_{\max }-\left(k_{\mathrm{on}}.Math.c_{\mathrm{analyte}}+k_{\mathrm{off}}\right).Math.\mathrm{tR}& \left(4\right)\end{array}$
The first derivative of the rising up-going part of the transformed Response (tR) with time

(89) $\frac{d\left(\mathrm{tR}\right)}{dt},$
was plotted against the transformed Response, tR. This yielded a straight line with a slope equal to −(k.sub.on.Math.c.sub.analyte+k.sub.off). Plotting this slope versus c.sub.analyte for the set of analyte concentrations (DA) yielded a graph from which k.sub.on and k.sub.off were calculated. The “Slope” versus DA concentration curve obtained is given in FIG. 12, which confirms that the model used for the sensorgram methodology is applicable. For the first time reliable molecular interaction data were obtained for the interaction of a biomolecule (the aptamer) with its target molecule, via a potentiometric sensor principle.

(90) Specific Detection with DA-Specific Aptamer

(91) To check the specificity of the DA-specific aptamer, Dopamine and three other basic drugs (ritodrine, lidocaine and promazine) were tested on the gelatin B-coated electrodes with and without the aptamer. These lipophilic cationic drugs have much better responses on PVC-based potentiometric sensors than DA. Sensors based on gelatin B were quite insensitive towards these three other drugs, there being no improvement in sensitivity for these compounds when the aptamer biorecognition element was coupled to the gelatin B, whereas DA showed a very clear increase in R.sub.max, as disclosed above. Table 1 gives the R.sub.max values (in mV) of 10.sup.−5M injections of different analyte molecules for different potentiometric electrodes.

(92) TABLE-US-00001 TABLE 1 Dopamine Ritodrine Lidocaine Promazine response response response response Coating in mV in mV in mV in mV Gelatin B 9.46 1.83 2.88 3.66 Gelatin B + DA- 92.86 1.57 1.57 2.44 specific aptamer

Example 3—Electrochemical CAP-Aptasensor Based on a SPE Gold Electrode

(93) Electrochemical measurements were recorded by a Autolab potentiostat controlled by NOVA 1.10 software package (Metrohm, The Netherlands). Morphological investigation of the electrode surface was done on a Fei Quanta 250 FEG Scanning Electron Microscope (SEM). The SPE was purchased from Metrohm and made of a gold working electrode, a carbon counter electrode and a silver reference electrode.

(94) Unless specified otherwise, the chemicals and materials are the same as in the previous examples.

(95) FIG. 13 schematically shows the construction of the electrochemical gelatin type B-based CAP-aptasensor. In the absence of CAP, the thiolated aptamer encapsulated in gelatin B is partially un-folded but linked to the gold surface by an Au—S bonding. When CAP is introduced to the modified SPE, the aptamer switches its structure to bind CAP bringing the redox active molecules proximate to the electrode surface resulting in an enhanced electron transfer.

(96) The first step is the electrochemical pre-treatment and biomodification of the gold screen printed electrode (SPE). Prior to immobilization of the thiolated DNA aptamer, a multiple-pulse amperometric pre-treatment of the gold surface is carried out in a stirred 0.5 mol L.sup.−1 H.sub.2SO.sub.4, 10 mmol L.sup.−1 KCl solution. The following triple-potential pulse sequence: −0.3 V for 3.0 s; 0.0 V for 3.0 s and +1.0 V for 1.5 s (15 cycles) was applied. The gold working electrode surface of SPE was then exposed to the mixture of aptamer (5 μM) and the solution of gelatin type B (5 w/v %) in tris buffer (pH 7.6). The percentage of the incorporation was 70:30 v/v % from the aptamer:gel mixture. Chemisorption is allowed to proceed (about 4 hours) while the electrodes are stored in a wet chamber to protect the solution from evaporation. The immobilization step is followed by addition of CAP solution (a 100 μL drop) on top of the modified-gold SPE for 25 minutes. Prior to the electrochemical measurement, the electrode was gently washed with 100 μL of tris buffer. Then, the differential pulse voltammetry is performed in tris buffer solution (pH 7.6).

(97) EIS measured data (FIG. 14) show that the electron transfer resistance increases in the following order: bare SPE (FIG. 14a), aptamer modified SPE (FIG. 14b) and aptamer/Gelatin B modified SPE (FIG. 14c). The increase in electron transfer resistance indicates that the aptamers are successfully immobilized on the electrode surface. In the case of a aptamer/GelB SPE, an additional barrier of negatively charged gelatin has increased the electron transfer resistance which results in a larger semi-circle. This demonstrates the successful immobilization of the aptamer on the SPE electrode and the blocking effect of the gelatin B layer against unspecific redox active molecules.

(98) In order to investigate the role of the gelatin B matrix in the efficiency of the aptasensor, differential pulse voltammetry (DPV) was selected as sensitive technique. FIG. 15 displays the differential pulse voltammograms of accumulated CAP (10.sup.−9 M) at the surface of bare gold SPE (curve a: 0.00 μA), Gelatin A modified SPE (curve b: 0.67 μA±0.02), Gelatin B modified SPE (curve c: 0.801±0.015 μA), aptamer modified SPE (curve d: 1.22±0.06 μA), aptamer/Gelatin A modified SPE (curve e: 0.86±0.04 μA) and aptamer/Gelatin B modified SPE (curve f: 2.71±0.09 μA) in tris buffer solution. The indicated currents reflect the peak current obtained in the voltammogram and can be explained as the irreversible reduction of the nitro group (NO.sub.2) present in CAP molecules, with formation of hydroxylamine (NHOH). As can be seen, there is no signal for CAP at a bare gold electrode. It means that no chemisorption takes place after 25 minutes accumulation of CAP at the surface of bare gold SPE (curve a). After immobilization of gel at SPE, the DPV signal appears for accumulated CAP (curve b and c). However, Gelatin B modified SPE shows a higher current signal than Gelatin A. Following the modification of the SPE by the aptamer, the current of DPV signal increased (curve d), confirming the ability of the synthesized aptamer to capture the target molecule. The combination of aptamer and Gelatin A had no positive effect on the sensor efficiency (curve e) while the incorporation of aptamer and Gelatin B showed a very good response toward the target molecule (curve f) at the potential at which we expect the reduction of the nitro group, i.e. −0.7 V. Also, there is a pre oxidation wave around −0.45V that shows the presence of intermediates in redox reactions of CAP.

(99) Dramatic increase in DPV height after mixing aptamer and Gelatin B is likely due to an increase in charge transfer kinetics resulting from the better reactivity of the aptamer towards the target. Due to the incorporation of the aptamer in the gelatin B matrix and its biocompatibility, most sites of the aptamer will remain active during the formation of the self-assembled monolayer from the thiolated aptamer. As the mixture of aptamer/gelatin B shows a significantly higher DPV signal than aptamer/gelatin A, the biocompatibility of gelatin B towards aptamers is expected to be better. Because of the physical interactions between the aptamer chains and gelatin (e.g. van der Waals forces and hydrogen bonds between amino acids), GelB is a good example of a physically cross-linked hydrogel. Therefore, the hydrophilic groups or domains which are hydrated make GelB a suitable matrix for the entrapment of the aptamer.

(100) To obtain the most sensitive results, parameters such as the kind (FIG. 16 A) and time (FIG. 16 B) of the CAP accumulation step were optimized for the electrochemical detection of CAP. Three approaches were investigated to accumulate CAP on the surface of the aptamer/GelB electrodes. First, the modified SPE was immersed in the CAP solution while the solution was stirred very fast (i). Secondly, the CAP solution was stirred slowly (ii). Thirdly the modified SPE was kept horizontally with a 100 μL of the CAP solution on top of it (wet drop, iii). The latter was proven to be the best to accumulate CAP because of the better interaction opportunity between CAP, aptamer, GelB and the electrode surface (FIG. 16A). Also, the time of accumulation for CAP on the surface of the aptamer/GelB modified SPE was investigated during 10, 15, 20, 25, 30 and 35 minutes. When accumulation time increased from 10 to 25 minutes, the signal of the accumulated CAP enhanced. After that, no obvious changes in the DPV reduction current of CAP were observed. Therefore, 25 minutes was selected as optimized time (FIG. 16 B).

(101) FIG. 17A depicts the diagnostic performance of the aptasensor. To study the role of gelatin B as matrix in function of the biosensor, the CAP reduction signal was investigated on the aptamer/gelatin B modified SPE. It can be seen that the peak current increases with an increase of the concentration of CAP (FIG. 17B). The peak current shows a linear relationship with the Log of concentration of CAP in the range from 0.10 to 10.0 pico mol L.sup.−1 (insert of FIG. 17B). The calculated detection limit is 2.09×10.sup.−14 mol L.sup.−1 based on the interpolation to the point where the current differs from the background current for three standard deviations calculated from the currents obtained for three different electrodes.

Example 4—Selective CAP Detection in a Milk Sample

(102) The assay of the target in a real sample was investigated by detecting CAP in a skimmed cow's milk sample with the SPE electrode of example 3. The standard addition method was employed to evaluate the applicability of the developed aptasensor. The increased reduction peak of CAP occurred in the expected potential range at aptamer/gelatin B modified SPE compared to the reduction current obtained at an aptamer immobilized electrode without a gelatin protective matrix, suggesting an enhanced sensitivity of the developed sensor. Recovery values shown in table 2, ranging between 82% and 95%, indicate the applicability of the developed aptasensor for CAP detection in real samples.

(103) TABLE-US-00002 TABLE 2 Added Detected Recovery R.S.D. Sample (M) (M) (%) (%) 1 10.sup.−9  9.50 + (0.36) × 10.sup.−10 95 3.78 2 10.sup.−10 8.60 + (0.30) × 10.sup.−11 86 3.48 3 10.sup.−11 8.23 + (0.26) × 10.sup.−12 82 3.15

(104) For selectivity study, thiamphenicol and florfenicol antibiotics with a structure similar to CAP were used. Results showed that they did not influence the performance of the aptasensor, suggesting a good selectivity of this aptasensor. Also for stability study, the impedance measurement was done after a CAP electrochemical detection. The same value was obtained, indicating that the self-assembled aptamer is quite stable.