Patent ID: 12228541

EXAMPLE 1—PREPARATION OF NANOMIPS VIA SOLID-PHASE SYNTHESIS

200 g of glass beads were incubated in boiling 4.0 M sodium hydroxide for 15 min, washed with deionised water and dried at 150° C. Activated glass beads were incubated for 10 hour in a solution 2% (v/v) of N-[3-(trimethoxysilyl)propyl]ethylenediamine (DAMO), and 0.33% of 1,2-bis(triethoxysilyl)ethane (BTSE) in toluene at 70° C., washed with acetone and dried at 150° C. Glass beads were immersed into 0.1 M MES buffer, pH 6.0 containing 0.01 M N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 3.6 mM N-hydroxysuccinimide (NHS), and mixed with 0.03 M dodecanedioic acid dissolved in DMF for 20 min. After that beads were washed with 50% DMF and acetone. The template was immobilised on the glass beads by incubation in 0.43 mM (S)-(−)-α-amino-γ-butyrolactone hydrobromide and 0.43 mM of sodium carbonate. Glass beads with immobilised template were washed with water and dried under vacuum. Monomer mixture was prepared by mixing 1.44 g of methacrylic acid (MAA), 1.62 g of ethylene glycol dimethacrylate (EGDMA), 1.62 g of trimethylolpropane trimethacrylate (TRIM), 0.37 g of N,N-diethyldithiocarbamic acid benzyl ester (DABE), 0.09 g of pentaerythritol-tetrakis-(3-mercaptopropionate) (PETMP), and 0.14 g of ferrocenylmethyl methacrylate (FMMA) and deoxygenating with nitrogen for 10 min. Glass beads with immobilised template (25 g) were degassed in vacuum for 20 min and coated with monomer mixture. Polymerisation was initiated by exposing the mixture to UV-light for 2 min (Philips model HB/171/A, 4×15 W/amps). After polymerisation, the crude of reaction was transferred into a solid phase extraction (SPE) cartridge fitted with a polyethylene frit (20 mm porosity) and washed with cold acetonitrile at 0° C. in order to remove monomers, residues and low affinity nanoparticles. The high affinity nanoMIPs were extracted by elution at 6° C.

In examples 2 to 4 the template was tetrahydrocannabinol (THC), morphine or N-butyryl-L-homoserine lactone (C4—HSL).

EXAMPLE 2—DLS ANALYSIS OF NANOMIPS

700 μL of nanoMIPs (0.02 mg/mL) in water, prepared according to example 1, which were imprinted with either THC (THC nanoMIPs) or morphine (morphine nanoMIPs) were mixed with 350 μL of water, and optionally 95 μM THC or morphine and briefly sonicated. The hydrodynamic size of nanoMIPs was measured by dynamic light scattering (DIS) with a ZetaSizer Nano ZS (Malvern Instruments Inc, UK). NanoMIPs conformational changes triggered by the analyte are shown in Table 1.

TABLE 1DLS results for nanoMIPs and nanoNIPs in solutionand loaded with THC and Morphine molecules.DiameterNanoparticle sample(nm)PDI*THC nanoMIPs307.3 ± 2.50.323THC nanoMIPs in the presence of THC379.2 ± 6.40.295THC nanoMIPs in the presence of morphine265.8 ± 3.20.261Morphine nanoMIPs195.4 ± 4.40.156Morphine nanoMIPs in the presence of THC179.3 ± 8.70.278Morphine nanoMIPs in the presence of morphine208.3 ± 4.50.363*Polydispersity index (PDI)

As shown in the table, the diameter of the nanoMIPs increases in the presence of the molecule with which it has been imprinted, i.e. the template molecule. This is shown schematically inFIGS.1and2.

EXAMPLE 3—IMMOBILIZATION OF NANOMIPS

NanoMIPs prepared according to example 1, were immobilised on gold electrodes using one of two methods.

(i) Gold electrodes (Drop-Sense gold electrodes DRP-250AT (aux.: Pt ref.: AgCl) were functionalized by incubation in 3 mM solution of crystamine in ethanol for 8 hours. Functionalised electrodes were incubated for 30 min in 100 μL solution containing 0.03 mg/mL of nanoMIPs, 0.4 M N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 0.1M N-hydroxysuccinimide (NHS) in 50 mM PBS.

(ii) NanoMIPs (3.6 mg/mL in acetonitrile) were mixed with 200 mg of gold polymer electrode paste (C2041206P2, Gwent Group U.K.) and spread over gold electrodes (Drop-Sense gold electrodes DRP-250AT (aux.: Pt ref.: AgCl). Electrodes were cured at 8° C. for 30 minutes.

EXAMPLE 4—ELECTROCHEMICAL MEASUREMENTS OF SMALL MOLECULES

NanoMIPs imprinted with C4-HSL were prepared according to example 1 and immobilised on gold electrodes by covalent attachment according to example 3.

All experiments were carried out using an Autolab11 Instruments, Netherland. Differential pulse voltammetry (DVP) was performed under the following experimental conditions: potential was recorded in the range of −0.4 to +0.8 V and the step potential and modulation amplitude at 50 mV. The current signal was measured at the redox potential of the redox marker in nanoMIPs (0.22 V vs AgCl).

DPV responses of nanoMIP-coated electrodes to C4-HSL solutions at concentrations between 0 and 50 μM are presented inFIG.3a. The nanoMIPs with anchored FMMA showed good response to the template with a sensitivity of 47.5 μA/μM (R2=0.998) and LOD of 0.13 μM. The sensor response of nanoMIPs-coated electrode to different homoserine lactones is shown inFIG.3b. The calibration plots demonstrate good selectivity of sensor for C4-HSL.

EXAMPLE 5—SYNTHESIS OF NANOMIPS IMPRINTED WITH TRYPSIN

For the polymerization mixture the following monomers were dissolved in 100 ml of water: 39 mg (0.214 mmol) of N-isopropylacrylamide (NIPAM), 6 mg (0.078 mmol) of N,N-methylene-bis-acrylamide (MBA), 2.2 μL (0.0224 mmol) of acrylic acid (AAc), 6 mg (0.0211 mmol) of ferrocenylmethyl methacrylate (FMAA) and 5.8 mg (0.0325 mmol) of N-(3-aminopropyl) methacrylamide hydrochloride (NAPMA). Additionally, 33 mg (0.264 mmol) of tert-butyl acrylamide (TBAM) dissolved in 2 mL ethanol were added to the aqueous mixture. The solution was sonicated for 5 min, and then purged with nitrogen for 30 min. 50 mL of the polymerization mixture were added to 60 g of trypsin-derivatized glass beads. Polymerization was initiated by addition of 0.5 mL of (60 mg/mL) ammonium persulfate and (30 μL/mL) tetramethylethylenediamine and continued at room temperature for 1 hour. Beads were decanted into solid phase extraction cartridge and washed with water (10 bead volumes, 50 mL). The SPE cartridge was then placed in water bath at 60° C. for 7 min and high affinity nanoMIPs eluted with water at 6° C. (5 bead volumes, 20 mL). The concentration of the nanoMIPs fractions was evaluated by freeze-drying of solution aliquots and weighing.

EXAMPLE 6—ELECTROCHEMICAL MEASUREMENTS OF TRYPSIN

All experiments were carried out using an Autolab11 Instruments, Netherland using a gold electrode prepared using nanoMIPs prepared according to example 5 and the method described in Example 3(i). Differential pulse voltammetry (DVP) was performed under the following experimental conditions: potential was recorded in the range of −0.4 to +0.8 V and the step potential and modulation amplitude at 50 mV. The current signal was measured at the redox potential of the redox marker in nanoMIPs (0.22 V vs AgCl).

DPV responses of nanoMIP-coated electrodes to trypsin solutions are presented inFIG.4a. The calibration curve shows the good sensitivity for trypsin 0.25 nM/μA (R2=0.998) and LOD of 0.15 μM. The responses from avidin (3.95×10−8nM, R2=0.897) and pepsin (8.8×10−8nM, R2=0.577) are negligible, six orders of magnitude lower compared to the trypsin as shown inFIG.4b. This shows that the system exhibits good sensitivity for trypsin.

EXAMPLE 7—SYNTHESIS OF NANOMIPS IMPRINTED WITH GLUCOSE

The monomer mixture was composed by 39 mg (0.214 mmol) of N-isopropylacrylamide (NIPAM), 6 mg (0.078 mmol) of N,N-methylene-bis-acrylamide (MBA), 33 mg (0.264 mmol) of tert-butyl acrylamide (TBAM), 2.2 μL (0.022 mmol) acrylic acid (AAc), 39 mg of N-iopropylacrylamide (NIPAM), 7 mg (0.028 mmol) of ferrocenylmethyl methacrylate (FMAA), 5.8 mg (0.033 mmol) of N-(3-aminopropyl) methacrylamide hydrochloride (NAPMA) and 30 mg (0.167 mmol) of glucose. The components were dissolved in 100 mL 2% ethanol in water (v/v), sonicated for 5 min, then degassed using nitrogen for 30 min. The polymerisation was initiated by the addition of a solution comprising 30 mg (0.132 mmol) ammonium persulfate (APS) and 30 μL (0.2 μmol) of N,N,N′,N′-tetramethylethane-1,2-diamine (TEMED) in 0.5 mL of water. The monomer mixture was allowed to polymerize at ambient temperature (25° C.) for 1 hour. Subsequently, MIP was washed with water 7 times in a centrifuge cartridge filter (10 kDa) to remove template and unreacted monomers. Next, the fractions of high affinity nanoparticles were collected.

EXAMPLE 8—ELECTROCHEMICAL MEASUREMENTS OF GLUCOSE

All experiments were carried out using an Autolab11 Instruments, Netherland using a gold electrode prepared using nanoMIPs prepared according to example 7 and the method described in Example 3(i). Differential pulse voltammetry (DVP) was performed under the following experimental conditions: potential was recorded in the range of −0.4 to +0.8 V and the step potential and modulation amplitude at 50 mV. The current signal was measured at the redox potential of the redox marker in nanoMIPs (0.22 V vs AgCl).

DPV responses of nanoMIP-coated electrodes to glucose solutions are presented inFIG.5a. For control measurements a polymer imprinted with dopamine, which was prepared using the method of example 7 but replacing the glucose with dopamine, was used. The control polymer known as non-specific imprinted polymer (NIP) reflects as expected a negligible response (24 times lower), seeFIG.5b. Additionally, the glucose sensor does not show high level of cross reactivity for fructose, maltose and lactose, seeFIG.6.

Conclusion

The inventors have shown that it is possible to synthesise MIPs imprinted with an analyte and comprising ferrocenylmethyl methacrylate (FMAA), a redox label. The inventors have shown that the diameter of the MIPs changes depending upon whether or not the analyte is present, as shown schematically inFIG.1.

The MIPs may be immobilised on a first electrode. The first electrode and a second electrode may then be placed in a solution and a potential difference applied across them. As the concentration of the analyte varies the volume of the MIPs also vary, changing the number of redox labels which contact the electrode, as shown inFIG.2. This in turn causes the current which passes between the first and second electrodes to vary, allowing the concentration of the analyte to be accurately calculated.

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