NANO-ENZYME CONTAINERS FOR TEST ELEMENTS

Abstract

The present invention relates to a test element for the detection of an analyte comprising an enzyme, wherein the enzyme is incorporated in a nanocapsule.

Claims

1. A diagnostic test element for the detection of an analyte comprising: an enzyme; and a silica nanocapsule substantially impermeable for the enzyme but permeable for an enzyme substrate, the enzyme being incorporated within the interior of the silica nanocapsule, the interior of the silica nanocapsule being free from a hydrophilic polymer.

2. The diagnostic test element of claim 1, wherein the nanocapsule comprises a polyaddition and/or polycondensation product of monomers formed at the interface in a water-in-oil miniemulsion comprising a continuous oil phase and a discontinuous aqueous phase dispersed therein, the enzyme being present in the aqueous phase.

3. The diagnostic test element of claim 2 wherein the silica nanocapsule comprises a polycondensation product of a tri- or tetra-alkoxy silane and a compound RSiX.sub.3 or R.sub.2SiX.sub.2, wherein X is OCH.sub.2, OCH.sub.2CH.sub.3 or halo, and R is a C.sub.3-C.sub.30 alkyl group or an optionally substituted C.sub.3-C.sub.30 alkyl group.

4. The diagnostic test element of claim 3 wherein the silica nanocapsule is a polycondensation product of a tetra-alkoxy silane with a compound RSiX.sub.3, wherein X is halo and R is an optionally substituted C.sub.3-C.sub.30 alkyl group.

5. The diagnostic test element of claim 4 wherein the halo is chloro.

6. The diagnostic test element of claim 1 wherein the nanocapsule has pores with a maximum size which is smaller than the size of the enzyme.

7. A method for detecting an analyte by an enzymatic reaction, comprising: providing a sample to be analyzed; contacting the sample with the test element of claim 1; and detecting an enzymatic reaction between the enzyme and the enzyme substrate.

8. The diagnostic test element of claim 1 wherein the interior of the silica nanocapsule is also free from a structuring surfactant.

9. The diagnostic test element of claim 8, wherein the nanocapsule comprises a polyaddition and/or polycondensation product of monomers formed at the interface in a water-in-oil miniemulsion comprising a continuous oil phase and a discontinuous aqueous phase dispersed therein, the enzyme being present in the aqueous phase.

10. The diagnostic test element of claim 9 wherein the silica nanocapsule comprises a polycondensation product of a tri- or tetra-alkoxy silane and a compound RSiX.sub.3 or R.sub.2SiX.sub.2, wherein X is OCH.sub.2, OCH.sub.2CH.sub.3 or halo, and R is a C.sub.3-C.sub.30 alkyl group or an optionally substituted C.sub.3-C.sub.30 alkyl group.

11. The diagnostic test element of claim 10 wherein the silica nanocapsule is a polycondensation product of a tetra-alkoxy silane with a compound RSiX.sub.3, wherein X is chloro and R is an optionally substituted C.sub.3-C.sub.30 alkyl group.

12. The diagnostic test element of claim 4 wherein the halo is chloro.

13. The diagnostic test element of claim 8 wherein the nanocapsule has pores with a maximum size which is smaller than the size of the enzyme.

14. A method for detecting an analyte by an enzymatic reaction, comprising: providing a sample to be analyzed; contacting the sample with the test element of claim 8; and detecting an enzymatic reaction between the enzyme and the enzyme substrate.

15. A diagnostic test element for the detection of an analyte comprising: an enzyme; and a silica nanocapsule being substantially impermeable for the enzyme but permeable for an enzyme substrate, the enzyme being incorporated within the silica nanocapsule, the nanocapsule being free from a structuring surfactant.

16. The diagnostic test element of claim 15, wherein the nanocapsule comprises a polyaddition and/or polycondensation product of monomers formed at the interface in a water-in-oil miniemulsion comprising a continuous oil phase and a discontinuous aqueous phase dispersed therein, the enzyme being present in the aqueous phase.

17. The diagnostic test element of claim 16 wherein the silica nanocapsule comprises a polycondensation product of a tri- or tetra-alkoxy silane and a compound RSiX.sub.3 or R.sub.2SiX.sub.2, wherein X is OCH.sub.2, OCH.sub.2CH.sub.3 or halo, and R is a C.sub.3-C.sub.30 alkyl group or an optionally substituted C.sub.3-C.sub.30 alkyl group,

18. The diagnostic test element of claim 17 wherein the silica nanocapsule is a polycondensation product of a tetra-alkoxy silane with a compound RSiX.sub.3, wherein X is chloro and R is an optionally substituted C.sub.3-C.sub.30 alkyl group.

19. The diagnostic test element of claim 15 wherein the nanocapsule has pores with a maximum size which is smaller than the size of the enzyme.

20. A method for detecting an analyte by an enzymatic reaction, comprising the steps: providing a sample to be analyzed; contacting the sample with the test element of claim 15; and detecting an enzymatic reaction between the enzyme and the enzyme substrate.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0041] FIG. 1: Schematic depiction of a permeable nanocapsule, having incorporated an enzyme molecule according to the invention.

[0042] A permeable polymeric shell (10), e.g. a poly(urethane/urea) shell or a silica shell, encapsulates a core medium (12), e.g. an aqueous buffered solution comprising an enzyme molecule (14), e.g. glucose dehydrogenase, and optionally a co-enzyme, e.g. NAD, or carba NAD (18). The polymeric shell (10) is permeable to enzyme substrate molecules (16), e.g. glucose.

[0043] FIGS. 2A-F: Scanning election microscope (SEM) images of nanocapsules prepared from 2-4-toluene diisocyanate (TDI) using different hydrophilic monomers: A: 1,6-hexanediol; B: lactic acid; C: 1,3-dihydroxyacetone; D: glycerol; E: poly(vinyl alcohol); F: hyaluronic acid.

[0044] FIGS. 3A-C: Synthesis and redispersion of GDH containing silica nanocapsules: [0045] A) Synthesis of nanocapsules in an inverse miniemulsion. [0046] B) Redispersion of nanocapsules in aqueous phase. [0047] C) SEM image of nanocapsules including a schematic depiction of shell structure.

[0048] FIGS. 4A-E: SEM images (obtained from cyclohexane phase) represent core-shell morphology of the silica nanocapsules (sample UPSI178).

[0049] FIG. 5: FTIR spectrum of lyophilised silica nanocapsules (UPSI178) with a molar ratio of OTS:TEOS (1:1.9) in cyclohexane phase.

[0050] FIGS. 6A-B: Solid state SiMAS NMR of lyophilised silica capsules prepared from a) OTS and TEOS, b) DTS and TEOS. The molar ratio between two precursors was kept constant at 1:1.9.

[0051] FIGS. 7A-B: .sup.29Si{.sup.1H} CP-MAS correlation spectra of sample UPSI162 probing (a) closest spatial proximities and (b) larger distances on the nm length scale.

[0052] FIGS. 8A-C: SEM images from silica nanocapsules prepared with different molar ratios of OTS:TEOS, namely a) 1:5.6, b) 1:3.7 and c) 1:1.9.

[0053] FIGS. 9A-B: .sup.13C-NMR of D.sub.2O hydrated thick shell of silica capsules containing GDH (OTS:TEOS was 1:1.9).

[0054] FIG. 10: Schematic depiction of the biocatalytic reaction between glucose and encapsulated GDH in the presence of methylene blue (MB).

[0055] FIG. 11: Decolorisation time of methylene blue (MB) dependent on the molar ratio of OTS:TEOS.

[0056] FIG. 12: Kinetic study of GDH activity in solution in the presence of silica nanocapsule (GDH is not encapsulated) with OTS/TEOS=1.1.9 and a GHD core concentration of 0.1 mg/ml.

[0057] FIG. 13: Kinetic study of encapsulated GDH activity in silica nanocapsule with OTS/TEOS=1:1.9 and a GHD core concentration of 0.1 mg/ml.

[0058] FIG. 14: Kinetic study of GDH activity for a silica nanocapsule with OTS/TEOS=1:1.9 and a GHD core concentration of 1 mg/ml.

[0059] FIG. 15: Kinetic study of GDH activity for a silica nanocapsule from TDI and 1,6-hexanediol.

[0060] FIG. 16A and FIG. 16B: Activity of free GDH and encapsulated inside silica shell after 12 weeks incubation at 35° C.; 85% relative humidity. For FIG. 16A, the Y-axis is labeled as follows: [0061] Lum Diff Ref (<=75‘-’ mg/dl, >75‘/’%) Code (0.0-600.0 mg/dl) P Spectr(7), SavGol(21, .sup.{circumflex over ( )}2), lraw,-o/lpre0.1s, a-Dyn.l (4.0s,0.0s, dl/l/dt>−1.0%/s), (base vector.6)), LMLog (xNrm wett.).

[0062] For FIG. 16B, the Y-axis is labeled as follows: [0063] Lum Diff Ref (<=75‘-’ mg/dl, >75‘/’%) Code (0.0-600.0mg/dl) P Spectr(7), SavGol(21, .sup.{circumflex over ( )}2), lraw,-o/lpre0.1s, a-Dyn.l (4.0s,0.0s,dl/l/dt>−1.0%/s), (base vector.7)), LMLog (xNrm wett.).

[0064] FIG. 17: Degree of mobility of free and encapsulated GDH in test strips. The obtained values for encapsulated GDH are lower compared to free GDH.

EXAMPLES

[0065] Materials

[0066] Glucose dehydrogenase (GDH), nicotinamide adenine dinucleotide (NAD.sup.+), carbo-NAD.sup.+, 2-amino-2(hydroxymethyl)1,3-propanediol (Tris) were provided from Roche, Germany. Sodium chloride (NaCl, analytical grade) and cyclohexane (HPLC) were supplied by VWR. Trichloro(octadecyl)silane (OTS, ≥90%) and tetraethoxysilane (TEOS, ≥99%) were purchased from Aldrich. Polyglycerol polyricinoleate (GRINDSTED® PGPR, Danisco A/S, Denmark) was applied as hydrophobic surfactant. Milli-Q water was used in all experiments. 1,6-hexanediol (Sigma Aldrich), 1,3-dihydroxyacetone (Acros), glycerol (Merck), poly(vinyl alcohol) (PVA) (M.sub.w=25000 g.Math.mol.sup.−1, Polysciences Inc), lactic acid (Sigma Aldrich) and hyaluronic acid (M.sub.w=140000 g.Math.mol.sup.−1, Fluka) were used as hydrophilic monomers for the polyurethane synthesis. The oil soluble block copolymer surfactant poly[(ethylene-co-butylene)-b-(ethylene oxide)], P(E/B-b-EO), consisting of a poly(ethylene-co-butylene) block (M.sub.w=3700 g.Math.mol.sup.−1) and a poly(ethylene oxide) block (M.sub.w=3600 g.Math.mol.sup.-1) was vacuum dried prior to use. The hydrophobic monomer toluene 2,4-diisocyanate (TDI, 98%) was purchased from Sigma Aldrich. Geropon®T77 was provided by ROCHE.

[0067] Characterization of Nanocapsules

[0068] The average size and the size distribution of the nanocapsules were measured by means of dynamic light scattering (DLS) with diluted dispersions (40 μl sample were diluted in 1 mL water) on a PSS Nicomp Particle Sizer 380 (Nicomp Particle Sizing Systems, USA) equipped with a detector at 90° scattering mode at 20° C.

[0069] Scanning electron microscopy (SEM) studies were done on a field emission microscope (LEO (Zeiss) 1530 Gemini, Oberkochen, Germany) working at an accelerating voltage of 170 V. Generally, the samples were prepared by diluting the capsule dispersion in cyclohexane or demineralized water (for redispersed samples) to about 0.01% solid content. Then one droplet of the sample was placed onto silica wafer and dried under ambient conditions over night. No additional contrast agent was applied. The solid content of the capsule dispersion was measured gravimetrically.

Example 1

Formation of Poly(Urethane/Urea) Nanocapsules with 1 mg/mL Enzyme (GDH)

[0070] Polyurethane nanocapsules were synthesized by a polyaddition reaction, whereas the reaction took place at the droplets' interface. The dispersed aqueous phase contained different monomers (see Table 1), which were mixed with 750 mg of enzyme solution. Enzyme solution was prepared as followed: 10 mg of GDH was dissolved in 1 mL of 0.1 M Tris buffer containing NaCl 0.2 M (pH 8.5) and incubated for 2 h at 4° C. The enzyme was diluted to 1 mg/mL with the diluting buffer (3.8 mM NAD.sup.+ or carba-NAD.sup.+ in Tris buffer).

[0071] The continuous oil phase contained the surfactant P(E/B-b-EO) (70 mg), which was dissolved in cyclohexane (6.0 g). Both phases were mixed together and the miniemulsion was obtained by ultrasonication. Ultrasonication procedure was applied for 3 min at 45% amplitude (20 s pulse, 10 s pause) using a Branson Sonifier W450-Digital under ice cooling in order to prevent evaporation of the solvent.

[0072] A clear solution consisting of cyclohexane (4 g), P(E/B-b-EO) (10 mg), and TDI (170 mg) was prepared and then added to the miniemulsion over 5 min at 25° C. The mixture was stirred for 24 h at 25° C. The size and size distribution was measured using dynamic light scattering (DLS), values see Table 1. The morphology was studied by scanning electron microscopy. The images are depicted in FIG. 1.

TABLE-US-00001 TABLE 1 Characterization of poly(urethane/urea) capsules obtained from different monomers. The monomer/TDI ratio was kept the same in runs from 1 to 4 as 1:1.5. Amount of monomer, Diameter, Run Monomer mg nm/STD, % 1 1,6-hexanediol 76 230/26 2 lactic acid 68 280/28 3 1,3-dihydroxyacetone 58 315/30 4 glycerol 140 295/30 5 poly(vinyl alcohol) 60 440/31 6 hyaluronic acid 30 360/30

Example 2

Formation of Poly(Urethane/Urea) Nanocapsules with 1, 0.1 and 0.01 m/mL of Enzyme (GDH)

[0073] Polyurethane nanocapsules were synthesized as described in Example 1. The dispersed aqueous phase contained different monomers (see Table 2), which were mixed with 750 mg of enzyme solution. Enzyme solution was prepared as followed: 10 mg of GDH was dissolved in 1 mL of 0.1 M Tris buffer containing NaCl 0.2 M (pH 8.5) and incubated for 2 h at 4° C. The enzyme was diluted to 1, 0.1 or 0.001 mg/mL with the diluting buffer (3.8 mM NAD.sup.+ or carba-NAD.sup.+ in Tris buffer).

[0074] The size and size distribution of the resulting capsules was measured using dynamic light scattering (DLS), values see Table 2.

TABLE-US-00002 TABLE 2 Characterization of poly(urethane/urea) capsules obtained from different monomers. The monomer/TDI ratio was kept the same in runs from 1 to 4 as 1:1.5. Diameter, nm/STD, % Amount of GDH concentration, monomer, mg per mL Run Monomer mg 1 0.1 0.001 1 1,6-hexanediol 76 230/26 235/27 230/27 2 lactic acid 68 280/28 270/29 280/28 3 1,3-dihydroxyacetone 58 315/30 325/30 360/31 4 glycerol 140 295/30 340/30 395/31 5 poly(vinyl alcohol) 60 440/31 475/31 525/33 6 hyaluronic acid 30 360/30 400/31 420/30

Example 3

Formation of Polyurethane Nanocapsules with 100, 200, 300 and 500 mg/mL of Enzyme (GDH)

[0075] Polyurethane nanocapsules were synthesized as described in Example 1. The dispersed aqueous phase contained a monomer, 1,6-hexanediol, (76 mg) and 750 mg of enzyme solution (100, 200, 300 or 500 mg/mL). Enzyme solution was prepared as followed: 100, 200, 300 or 500 mg of GDH was dissolved in 1 mL of 0.1 M Tris buffer containing NaCl 0.2 M (pH 8.5) and incubated for 2 h at 4° C.

[0076] The average size, size distribution and the enzyme activity of the obtained capsules are summarized in Table 3.

TABLE-US-00003 TABLE 3 Characterization of poly(urethane/urea) capsules obtained from 1,6-hexanediol and TDI. Concentration of GDH activity GDH solution for (KU/g capsules preparation Diameter, lyophilised Sample (mg/mL) nm/STD, % capsules) UPGDH1 100 202/43 14.7 UPGDH2 200 310/31 20.3 UPGDH3 300 200/42 30.1 UPGDH4 500 120/43 45.5

Example 4

Formation of Silica Nanocapsules with 1 and 0.1 mg/mL of Enzyme (GDH)

[0077] A heterophase mixture comprising 0.65 g of enzyme solution (1 or 0.1 mg/mL) as a dispersed phase and 6.25 g of cyclohexane containing 55 mg of the hydrophobic surfactant PGPR as a continuous phase was prepared. Enzyme solution was prepared as followed: 10 mg of GDH was dissolved in 1 mL of 0.1 M Tris buffer containing NaCl 0.2 M (pH 8.5) and incubated for 2 h at 4° C. The enzyme was diluted to 1 or 0.1 mg/mL with the diluting buffer (3.8 mM NAD.sup.+ or carba-NAD.sup.+ in Tris buffer).

[0078] The inverse miniemulsion was obtained by ultrasonication of the mixture at 45% amplitude (20 s pulse and 10 s pause) for 3 min under an ice cooling. A known amount of OTS/TEOS mixture (1:1.9 molar ratio) and 10 mg of PGPR dissolved in 2 g of cyclohexane were added drop-wise into the miniemulsion. The hydrolysis and co-condensation of the silane mixture took place at the droplets interface with subsequent formation of the silica shell. The reaction was carried out at room temperature overnight.

[0079] A schematic depiction of capsule synthesis and subsequent redispersion in an aqueous phase is shown in FIGS. 3A and 3B. A SEM image of silica nanocapsules including a schematic depiction of the shell structure is shown in FIG. 3C.

Example 5

Formation of Silica Nanocapsules with 300 mg/mL of Enzyme (GDH) (sample UPSI178)

[0080] Silica nanocapsules with 300 ng/mL GDH were prepared substantially as described in Example 4. Enzyme solution was prepared as follows: 300 mg of GDH were dissolved in 1 mL of PBS buffer (pH 8.5) and the solution was incubated for 2 h at 4° C.

[0081] The inverse miniemulsion was obtained by ultrasonication of the mixture at 45% amplitude (20 s pulse and 10 s pause) for 3 min under an ice cooling. A known amount of OTS/TEOS mixture (1:1.9 molar ratio) and 10 mg of PGPR dissolved in 2 g of cyclohexane were added drop-wise into the miniemulsion. The hydrolysis and co-condensation of the silane mixture took place at the droplets interface with subsequent formation of the silica shell. The reaction was carried out at room temperature overnight.

[0082] The obtained nanocapsules in the cyclohexane phase were two times centrifuged at 2000 rpm for 20 min to remove the oil-soluble surfactant (PGPR) from the suspension. The washed nanocapsules were redispersed in 0.5 wt % Geropon®T77 solution and afterwards freeze-dried. The lyophilised nanocapsules were purified by repetitive centrifugation in water to separate not-encapsulated enzyme from the sample. The washed capsules were redispersed in 0.5 wt % Geropon®T77 before being freeze-dried again and stored as a powder until the use. The morphology was studied by scanning electron microscopy. The images are shown in FIG. 4.

[0083] The inorganic content of 10.1 wt % with respect to all ingredients (from the cyclohexane phase) was determined by thermogravimetric analysis (TGA).

Example 6

Formation of Silica Nanocapsules with Different Molar Ratios of OTS:TEOS and 1 mg/mL of Enzyme (GDH)

[0084] Silica nanocapsules with different molar ratios of OTS:TEOS were prepared.

[0085] An inverse miniemulsion was obtained as described in Example 4. Different mixtures of OTS/TEOS (see Table 4) and 10 mg of PGPR dissolved in 2 g of cyclohexane were added drop-wise into the miniemulsion. The hydrolysis and co-condensation of the silane mixture took place at the droplets interface with subsequent formation of the silica shell. The reaction was carried out at room temperature overnight.

TABLE-US-00004 TABLE 4 Characterization of silica capsules obtained with different molar ratio of silica precursors (OTS:TEOS). Average particle Inorganic content (%) OTS:TEOS size Theoretical TGA at Samples (molar ratio) (nm) R.sub.w values 900° C. UPSI122 1:5.6 225 37 29 16 UPSI123 1:3.7 225 52 28 25 UPSI124 1:1.9 256 89 27 24 (30 μL) UPSI125 1:1.9 294 51 41 20 (60 μL)

Example 7

Silica Nanocapsules with Different Alkyl Chain Length of Alkyltrichlorosilane (Molar Ratio of RSiCl.SUB.3 .to TEOS Was Fixed at 1:1.86) and 1 mg/mL of Enzyme (GDH)

[0086] Silica nanocapsules with different alkyltrichlorosilanes, i.e. octadecyltrichlorosilane (OTS), dodecyltrichlorosilane (DTS) and hexyltrichlorosilane (HTS) were prepared.

[0087] An inverse miniemulsion was obtained as described in Example 4. A known amount of RSiCl.sub.3/TEOS mixture (see Table 5) (molar ratio of RSiCl.sub.3 to TEOS was fixed at 1:1.9) and 10 mg of PGPR dissolved in 2 g of cyclohexane were added drop-wise into the miniemulsion. The hydrolysis and co-condensation of the silane mixture took place at the droplets interface with subsequent formation of the silica shell. The reaction was preceded at room temperature overnight.

TABLE-US-00005 TABLE 5 Characterization of silica capsules obtained with different alkyl chain length of alkyltrichlorosilane (RSiCl.sub.3). Average particle Inorganic content (%) Alkyl chain length size Theoretical TGA at Sample of RSiCl.sub.3 (nm) calculation 900° C. USPI141 C.sub.18H.sub.27 (OTS) 225 29 26 UPSI162 C.sub.12H.sub.25 (DTS) 222 33 14 UPSI163 C.sub.6H.sub.13 (HTS) 222 36 11

Example 8

Characterization of Silica Nanocapsules Containing GDH

[0088] FTIR was employed to characterize the chemical composition of the silica capsules (FIG. 5). The peaks at 3450 cm.sup.−1 and at 940 cm.sup.−1 correspond to hydrogen bonded —OH stretching of silanol group and of Si—OH stretching, respectively. A high intensity band localized at 1064 cm.sup.−1 is attributed to Si—O—Si stretching with a shoulder at 1150 cm.sup.−1 from Si—C stretching (alkylsilane moiety). The peaks centred at 2850 and 2900 cm.sup.−1 correspond to CH stretching of alkyl moieties. The small peak at 815 cm.sup.−1 appears to be asymmetric flexion of the Si—O. The presence of encapsulated enzyme (GDH) in the samples could be confirmed by the presence of peaks at 3250, 1602 and 1722 cm.sup.−1 which are characteristic for —NH and amide stretching.

[0089] .sup.29Si MAS NMR in the solid state has been used to determine the degree of condensation and the local structure in terms of Q.sup.(n) and T.sup.(n) sites of the obtained silica material. To obtain quantitative results for the different Q.sup.(n) and T.sup.(n) sites in the materials, direct excitation spectra and MAS conditions have been recorded with a small excitation angle (˜20°), 15 s relaxation delays and high power .sup.1H decoupling. The results are listed in Table 6. Remarkably, the higher yield obtained for the trichloro silanes with longer alkyl chains does not lead to a better degree of condensation of the silica network, which would be reflected in a higher content of higher order Q groups, as the ideal silica network would consist of Q.sup.(4) groups only. In fact, the condensation in sample UPSI162 seems to be more complete compared to that of UPSI141. The .sup.29Si{.sup.1H} CP-MAS correlation spectrum shown in FIG. 6 demonstrates that the aliphatic protons of the organic substituent of T.sup.(n) groups in the material correlate only with the .sup.29Si NMR signals around −60 ppm, assigned to T groups, which Q.sup.(3) groups at −100 ppm correlate with a very broad OH proton signal centred around 5 ppm in the proton dimension. When a spin diffusion delay is introduced in the correlation experiment, correlations on larger length scales can be probed.

TABLE-US-00006 TABLE 6 Results of solid state .sup.29Si MAS NMR analysis. Alkyl Silica structure: chain Average Ratio of silaxane length of diameter units (%) Sample RSiCl.sub.3 (nm) T.sup.2 T.sup.3 Q.sup.2 Q.sup.3 Q.sup.4 UPSI124 C.sub.18H.sub.37 225 16 20 8 38 18 (OTS) UPSI162 C.sub.12H.sub.26 222  7 19 1 34 39 (DTS)

[0090] Q.sup.n: (Si(OSi).sub.n(OH).sub.4−n (n=0-4); T.sup.n: (R(OSi).sub.n(OH).sub.3−n (n=0-3)

[0091] The spectrum shown in FIG. 7b has been recorded with a diffusion delay of 10 ms, which corresponds to distances below 1 nm in a relatively dilute proton spin bath like our silica material. However a well-defined distance is very difficult to estimate, due to the heterogeneities of the .sup.1H spin with a high .sup.1H density at the aliphatic chains of the TH groups and a low .sup.1H density at the Q.sup.(n) sites, which experimentally leads to the finding that .sup.1H polarization diffuses from the T.sup.(n) groups to the Q.sup.(n) but the polarization of the OH groups at Q.sup.(2) and Q.sup.(3) sites cannot be observed at T.sup.(n) sites. This experiment clearly demonstrates that T.sup.(n) and Q.sup.(n) sites are in close spatial proximity in the silica material and in particular from the missing correlation signal between aliphatic protons and Q.sup.(n) sites in FIG. 7a any incorporation of surfactant into the silica material can be excluded.

[0092] An increasing molar ratio of alkyltrichlorosilane, e.g. OTS to tetraalkoxysilane, e.g. TEOS gave an increased shell thickness as shown in FIG. 8.

Example 9

Biocatalytic Properties of Encapsulated GDH

[0093] The crucial factors, which determine the biocatalytic properties of the encapsulated GDH are related to the diffusion limitation of glucose through the shell and enzyme stability.

[0094] The enzyme mobility in silica capsules was investigated by .sup.13C CP-MAS NMR. The presence of the Tris buffer and GDH was confirmed by the peaks at 60 and 65 ppm denoted as C—O and 170 ppm attributed to carbonyl groups. The overall decrease of the signal intensity in the CP-MAS spectrum of the hydrated sample, and in particular the vanishing of the carbonyl signal due to the higher molecular mobility in the hydrated state was clearly observed (FIG. 9). This could be a strong evident to mention that the enzyme is trapped in the dried capsules, which could be hydrated to enable enzyme to mobile in the small confinement.

[0095] The enzymatic activity of GDH encapsulated in silica nanocapsules was determined by the colorimetric analysis based on a reduction of methylene blue (MB) in the presence of NADH (Yudkin, Biochem. J. 28 (1934), 1454) which is produced during the glucose oxidation (FIG. 10). The reactions are triggered when the glucose molecules are able to diffuse inside the capsules containing GDH.

[0096] According to the obtained data all nanocapsules showed enzymatic activity. The decolorization time for all studied capsules was higher than that for the reference sample (silica capsules physically mixed with free GDH solution), indicating that the size of the pores is smaller than the size of the enzyme (which is 4.1 nm in radius determined from DLS). It could be also seen that the decolorization time is lower (the permeability is faster) for the silica capsules having less shell material. After encapsulation, the enzyme GDH still remains active, having about 70% of the initial activity.

[0097] The decolorisation time was dependent from the molar ratio of the alkyltrichlorosilane (e.g. OTS) to TEOS as shown in FIG. 11.

[0098] In FIG. 12 a kinetic study of the GDH activity measured by methylene blue decolorisation for a silica nanocapsule having a 15 nm thick shell (OTS/TEOS=1/1.9) and a core GDH concentration of 0.1 mg/ml is shown. The decolorisation rate for the reference sample (free enzyme) was as fast as the decolorisation rate with encapsulated enzyme. The observed time difference was based on a delay for the encapsulated enzymes caused by the time required for a diffusion of glucose through the capsule shell (diffusion time).

[0099] In FIG. 13 a kinetic study for a thin shell nanocapsule (molar ratio OTS:TEOS=1.9) with a core GDH concentration of 0.1 mg/ml is shown. In this case, no delay is found, i.e. a rapid diffusion through the thin shell takes place.

[0100] When increasing the enzyme concentration, the reaction rate increases as shown in FIG. 14 for a thin shell silica nanocapsule (molar ratio OTS:TEOS=1:1.9) and a GDH concentration in the core of 1 mg/ml.

[0101] A kinetic study of the enzymatic reaction in polyurethane capsules (comonomer 1,6-hexanediol) is shown in FIG. 15. After freeze-drying and resuspending, the capsules had the same response time as the reference, i.e. unencapsulated GDH.

Example 10

Stability, Activity and Mobility Test for Enzyme Activity in Dry Layer Chemistry

[0102] To show the application of GDH containing silica nanocapsules as glucose biosensors, the stability, activity and mobility tests in dry layer films were performed. The capsules containing 300 mg/ml GDH were synthesized for these studies using a molar ratio of OTS:TEOS of 1:1.9. The average particle size was 275±46 nm, the silica content was 13.8% and the specific activity 22 KU/g.

[0103] Test strips were manufactured comprising a first enzyme layer with either free GDH (280 KU/m.sup.2) or encapsulated GDH (86 KU/m.sup.2) and a second coating layer. The resulting test strips were incubated at 35° C. and 85% relative humidity. After 1, 6 and 12 weeks the total activity was determined. As can be seen in FIG. 16, the encapsulated enzyme shows higher stability in total activity than free GDH. Whereas free GDH shows 50-60% deviation after 6 weeks, the encapsulated GDH only shows 20-30% deviation. Thus, the encapsulated enzyme has a significantly higher functional stability than free enzyme, particularly when stored under warm and humid conditions.

[0104] For the elution of enzyme the test strip was treated for 10 minutes with ultrasound on ice in Tris buffer (0.1 M Tris; 0.2 M NaCl; 0.1% (w/v) albumin at pH 8.5). The activity was measured in the same Tris buffer system and the working solution in the cuvette contained additional 0.142 M glucose and 1.2 mM NAD. The absorption of the product NADH was detected at 340 nm in a spectrophotometer and the activity was calculated using the extinction coefficient of 6.3 [l mmol.sup.−1 cm.sup.−1]. The obtained data reveal the activity of 22.9 KU/capsule, which corresponds to 86 KU/m.sup.2. For comparison, the activity of free GDH was 280 KU/m.sup.2.

[0105] To characterize the enzyme mobility in the layer, the activity of supernatant obtained after washing the test strip was analyzed. The degree of mobility was defined as the activity of the supernatant in comparison to the total activity (FIG. 17).

CONCLUSION

[0106] The synthesis of silica nanocapsules with the average diameter of preferably 200-350 nm and containing active nicotinamide adenine dinucleotide (NAD.sup.+/NADH) dependent GDH inside the aqueous core was achieved in a single-step inverse miniemulsion process by controlling restricted interfacial hydrolysis and co-condensation of silica precursors. The properties of the encapsulated GDH in silica capsules in terms of encapsulation efficiency, wettability, and mobility of the enzyme in the silica capsules of the enzyme has been investigated. The permeability of the silica shell enables the enzyme to be wet and mobile in the hydrated state. In addition, the encapsulated enzyme shows high activity and similar to free enzyme stability in dry layer strip tests. This engineered silica capsules provide a new horizon for the developing of glucose sensitive biosensors.

[0107] Polyurethane/urea nanocapsules with encapsulated GDH were also investigated. The enzyme encapsulated therein shows high activity and thus is suitable for application to diagnostic test strips.