WO2015185504A1 - Biocapteurs à cristaux liquides - Google Patents

Biocapteurs à cristaux liquides Download PDF

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Publication number
WO2015185504A1
WO2015185504A1 PCT/EP2015/062151 EP2015062151W WO2015185504A1 WO 2015185504 A1 WO2015185504 A1 WO 2015185504A1 EP 2015062151 W EP2015062151 W EP 2015062151W WO 2015185504 A1 WO2015185504 A1 WO 2015185504A1
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product
mesophase
reactant
axis
substrate
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PCT/EP2015/062151
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English (en)
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Raffaele Mezzenga
Jijo VALLOORAN
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Eth Zurich
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G01N21/23—Bi-refringence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/02—Mechanical
    • G01N2201/022—Casings
    • G01N2201/0221—Portable; cableless; compact; hand-held

Definitions

  • Enzyme-linked immunosorbent assays are possibly the most widely used assays due to their relative simplicity, although they typically require several hours for the detection process.
  • ELISA is an example of an assay based on the conversion of a substrate to a product, which is of vital importance in the detection of pathological microorganisms, catalytic antibodies, and screening libraries of potential drugs and inhibitors.
  • Biosensors generally use a biological receptor compound such as an enzyme, nucleic acid or antibody to produce a physical or physico-chemical change in the environment, which can be used to detect the analytes. They allow the detection of a broad spectrum of analytes in complex sample matrices, and have shown great promise in areas such as food and water analysis, medical diagnostics and bio-threat monitoring.
  • a biological receptor compound such as an enzyme, nucleic acid or antibody
  • lipid-based surfactants When mixed with water, lipid-based surfactants self-assemble into different liquid crystalline symmetries such as the lamellar phase, inverted hexagonal phase and the bicontinuous cubic phase.
  • liquid crystalline mesophases have been used in controlled release and delivery systems and find diverse applications in food, cosmetics and pharmaceuticals. They also provide an excellent matrix for the entrapment of proteins and enzymes due to their biocompatibility, the presence of both hydrophilic and hydrophobic environments and their thermodynamic stability in excess water.
  • the objective of the present invention is to provide a novel method of detecting analytes or microorganisms. This objective is obtained by the subject matter of the independent claims.
  • the present invention seeks to improve upon the prior art detection methods by providing a novel biosensing platform. This is based on the birefringence evolution from a reaction product within lipid-based lyotropic liquid crystal cubic phases.
  • ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (CAS number 30931 -67-0).
  • TMB 3,3',5,5'-tetramethylbenzidine (CAS number: 54827-17-7).
  • OPD is o-phenylenediamine dihydrochloride (CAS number: 95-54-5).
  • crossed polarizer is defined as two linear polarizers that are placed in parallel alignment, with their polarizing axes in orthogonal arrangement with respect to each other.
  • the second polarizer is also referred to as analyzer.
  • chromogenic substrate refers to a chemical substance capable of conversion into a pigment or dye.
  • lipid based surfactant refers to an amphiphilic substance, wherein the hydrophobic part comprises a fatty acid and the hydrophilic part comprises glycerol.
  • a method for visually detecting a reaction from a reactant to a product comprises the steps of:
  • a birefringence value of the mesophase is measured.
  • insoluble in the context of the present specification signifies that a compound designated insoluble is present, under conditions of thermodynamical equilibrium, to a significant degree as a solid rather than in solubilized form. While minor amounts of the predominantly solid compound may be present in the liquid phase, in the context of the invention the important aspect is that at least a significant part of the compound is not solubilized.
  • the liquid crystal is in lamellar phase, inverted hexagonal phase or bicontinuous cubic mesophase.
  • a method for detecting an analyte comprising the steps of: i. providing the analyte in a bicontinuous cubic mesophase,
  • a birefringence value birefringence of the mesophase is measured.
  • the birefringence of the mesophase is measured continuously.
  • the lyotropic liquid crystal in bicontinuous cubic mesophase comprises water and a lipid-based surfactant or phospholipid.
  • the lyotropic liquid crystal in bicontinuous cubic mesophase contains 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45% of water.
  • the lyotropic liquid crystal in bicontinuous cubic mesophase contains from 20% to 35% of water.
  • the lipid-based surfactant is selected from monolinolein and mono-olein.
  • the lyotropic liquid crystal in bicontinuous cubic mesophase comprises monolinolein and contains from 20% to 35% of water.
  • the lyotropic liquid crystal in bicontinuous cubic mesophase comprises water and phytantriol (CAS no. 74563-64-7).
  • the reactant is selected from ABTS, TMB and OPD.
  • the reactant is a chromogenic substrate characterized in that its horseradish peroxidase (HRP) reaction product has low solubility in the cubic mesophase.
  • the product is selected from 3, 3', 5,5'- tetramethylbenzidine diimine, 2,3-diaminophenazine and the horseradish peroxidase reaction product of ABTS.
  • reaction step, the first cascade step, and/or the second cascade step is catalysed by an enzyme.
  • the enzyme is immobilized or entrapped within the highly viscous mesophase.
  • the enzyme is selected from a peroxidase, glucose oxidase or a cholesterol oxidase.
  • the peroxidase is linked to an antibody.
  • the peroxidase is horseradish peroxidase.
  • the analyte is by non-limiting example glucose, cholesterol, a microorganism, particularly a prokaryotic microorganism, more particularly a bacterium, or a virus, particularly a retrovirus, more particularly HIV, and said substrate is hydrogen peroxide.
  • the birefringence is measured during all steps of said method.
  • FIG. 8 is a schematic constructional drawing of the apparatus 10 according to a certain embodiment.
  • the apparatus 10 includes a volume 1 1 that is delimited by a first 12 and a second transparent optical element 13.
  • the first 12 and the second transparent optical element 13 are orthogonal to an axis 15 passing through the volume 1 1.
  • the first transparent optical element 12 is characterized by a first axis of polarization 16.
  • the second transparent optical element 13 is characterized by a second axis of polarization 17 rotated by 90° around the axis 15 in relation to the first axis of polarization.
  • This aspect of the invention is characterized in that the volume 1 1 comprises a bicontinuous cubic mesophase.
  • first and second transparent optical elements are linear polarizers that are placed in parallel alignment, with their polarizing axes in orthogonal arrangement with respect to each other.
  • This arrangement constitutes crossed polarizers that block light traversing parallel to axis 15. Any content of volume 1 1 that induces birefringence would allow some light to pass the second polarizer.
  • the apparatus comprises a second axis of polarization that is rotated around axis 15 in relation to the first axis of polarization by an angle of 50°, 60°, 70°, 80°, 85°, 90°, 95°, 100°, 1 10°, 120° or 130°.
  • the apparatus comprises a light source 18 being designed to emit visible light along axis 15.
  • a diagram illustrates the apparatus 9 according to some embodiments.
  • the apparatus 10 includes a diffusor 19, a main 22 and a contact switch 24, a battery 21 , a constant current supply 20, a light source 18 and a ventilator 23.
  • the second transparent optical element 13 is integrated into a hinge operated lid 25.
  • the bicontinuous cubic mesophase in volume 1 1 comprises a reactant and a substrate that is converted with the reactant into an insoluble product (insoluble in the liquid phases present in the reaction) in a reaction step.
  • the bicontinuous cubic mesophase in volume 1 1 comprises an analyte and an enzyme converting the analyte into a substrate in a first cascade step and a reactant that is converted with the substrate into an insoluble product (insoluble in the liquid phases present in the reaction) in a second cascade step.
  • the bicontinuous cubic mesophase comprises water and monolinolein.
  • the reactant is selected from ABTS, TMB, and OPD.
  • the product is selected from the horseradish peroxidase reaction product of ABTS, 3,3',5,5'-tetramethylbenzidine diimine and 2,3-diaminophenazine.
  • reaction step, the first cascade step, and/or the second cascade step is catalysed by a peroxidase, a glucose oxidase or a cholesterol oxidase.
  • the peroxidase is linked to an antibody.
  • the substrate is hydrogen peroxide and the analyte is glucose or cholesterol.
  • Fig. 1 shows a cross-polarized optical microscopy images of the cubic phase before (a) and after 3h of enzymatic reaction using the substrate ABTS (b), TMB (c) and OPD (d). Scale bar corresponds to 50pm. Insets show the visual appearance of each sample.
  • Fig. 2 Enzymatic conversion of ABTS into a colored product undergoing crystallization within the cubic phase, (a) Typical kinetic evolution followed by UV-vis absorption, (b) Small and (c) wide angle X-ray scattering spectra of the cubic phase before and after the enzymatic reaction.
  • Fig. 3 Illustration of birefringence development in cubic phases immobilizing HRP.
  • (a-j) POM images of the cubic phase taken every two minutes of the enzymatic reaction with 9.1 18 mM ABTS as substrate. Scale bar corresponds to 50 pm.
  • (k) Plot of the normalized birefringence intensity versus time arising from the conversion of 9.1 18 mM ABTS.
  • (I) Plot of the normalized birefringence intensity versus time at different ABTS substrate concentrations. ⁇ Illustration of glucose and cholesterol biosensors based on birefringence detection.
  • Fig. 5 Schematic representation of birefringent ELISA for pathogens detection.
  • Fig. 6 (a) Light microscopy image of immobilized E.coli in the cubic phase. Inset shows a fluorescence microscopy image, (b) POM image after the birefringent ELISA at 37°C. Inset shows a POM image of the control sample (no E.coli). (c) Photographs of the samples (top two images) and their visualization by the birefringent ELISA in the cross-polarized filter device (bottom two images) at 37°C. (d) Photograph of the portable birefringent ELISA device.
  • Fig. 7 Polarized optical microscopy image of inverted hexagonal phase before (left image) and after (right image) the enzymatic reaction.
  • FIG. 8 Schematic diagram of the device.
  • Apparatus 10 includes a volume 1 1 that is delimited by a first 12 and a second transparent optical element 13.
  • the first 12 and the second transparent optical element 13 are orthogonal to an axis 15 passing through the volume 1 1 .
  • the first transparent optical element 12 is characterized by a first axis of polarization 16.
  • the second transparent optical element 13 is characterized by a second axis of polarization 17 rotated by 90° around the axis 15 in relation to the first axis of polarization.
  • Fig. 9 Schematic diagram of the device in accordance with certain embodiments.
  • Fig. 10 Top view of the device where the birefringence is visible.
  • Fig. 1 1 Proposed device for the quantification of analytes.
  • Fig. 12 Illustration of HIV detection,
  • Inset shows the POM image of a control sample (cubic phase made of only human plasma),
  • Inset shows the POM image of heat treated sample after the birefringent ELISA, at 37 °C.
  • Fig. 13 Illustration of P. falciparum detection in blood samples, (a) Cross polarized optical microscopy image of a cubic mesophase produced using blood infected with P. falciparum parasites. The inset shows the negative control from a mesophase produced with uninfected blood, (b) Photograph of two reservoirs in a 96-well plate, visually observed through the cross-polarized device, containing: (top image) a cubic mesophase produced using infected blood and (bottom image) the control negative sample, (c) Photograph of two reservoirs in a 96-well plate, observed through the cross-polarized device, containing: (top image) a cubic mesophase produced using a buffer with extracted hemozoin and (bottom image) the control negative sample with uncontaminated buffer.
  • HRP horseradish peroxidase
  • the concept of encapsulating the enzyme Horseradish peroxidase (HRP) within a bicontinuous cubic phase, while preserving its functionality, is demonstrated herein by the conversion of suitable substrates into colored products.
  • the unique structure of the cubic phase with its two continuous water channels separated by a lipid bilayer, provides a unique platform for the immobilization of the enzyme in its active form, while allowing a fast diffusion of the substrate within the matrix.
  • the converted product induces birefringence in the system due to its crystallization, providing an efficient way to monitor the real-time enzymatic reaction when observed between crossed polarizers.
  • the inset of Figure 1 a shows a picture of a bicontinuous cubic phase sample just at the beginning of the enzymatic reaction. Over time, the peroxidase enzymatic reaction in presence of H 2 0 2 produced typical green (inset of Fig 1 b), blue (inset of Fig 1 c) and orange (inset of Fig 1 d) colored products within the cubic phase, when run separately with three different organic substrates: 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 3,3,5,5-tetramethylbenzidine (TMB) and o-phenylenediamine dihydrochloride (OPD), respectively.
  • ABTS 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)
  • TMB 3,3,5,5-tetramethylbenzidine
  • OPD o-phenylenediamine dihydrochloride
  • the SAXS spectra of the cubic phase before and after the enzymatic reaction show that the cubic phase remains essentially unchanged throughout the enzymatic reaction and confirm that the evolving birefringence does not arise from changes in the mesophase.
  • the slightly increased lattice parameter of the cubic phase after the enzymatic reaction may be due to the additional H 2 0 molecules formed from the conversion of H 2 0 2 during the enzymatic reaction, or from the simultaneous decrease in the molecules of substrate dissolved in the water phase, caused by their simultaneous crystallization.
  • the GOD catalyzes the conversion of ⁇ -D-glucose and oxygen to D-glucono-1 ,5-lactone and hydrogen peroxide.
  • the hydrogen peroxide arising from the first reaction then oxidizes ABTS in the presence of HRP, which in turn results in the formation of birefringence (Fig 4a-f).
  • Fig 4a-f the overall birefringence intensity generated by the oxidized product after 12 min reaction at 37 °C, shows a linear increase with glucose concentration in the range 3-8 mM.
  • birefringence from an enzymatic cascade reaction is general enough to be scalable and adaptable to the detection of other molecules: Therefore its generality is demonstrated by applying the same concept on another model analyte, this time purely hydrophobic in nature: cholesterol.
  • the measurement of cholesterol is of great importance in clinical applications because an abnormal amount of cholesterol in blood can cause clinical disorders such as heart disease, hypertension, arteriosclerosis and coronary artery disease.
  • Cholesterol oxidase (ChOD) and HRP were used to perform the bi-enzymatic cascade reaction and the birefringence developed at 37 °C showed again a perfectly linear correlation with the cholesterol concentration in the range 10-30 mM, as shown in fig 4h.
  • the methodology proposed here has the potential to be adapted to the detection of virtually any pathological microorganisms, making birefringent ELISA a versatile tool for the rapid, facile and inexpensive detection of analytes and pathogens in a broad range of biotechnological fields.
  • the method of the invention can be also utilized for the detection of viruses. Detection of HIV is a representative case of immediate relevance. Rapid detection of p24 antigen can be exploited for the early diagnosis of HIV infection in a cost-effective way, which is of prime importance in poor regions where expensive nucleic acid-based tests cannot be afforded. To this end, the current inventors designed a birefringent ELISA assay for the detection and quantification of model analyte HIV-1 capsid antigen p24 in human serum, in 1 h.
  • the aqueous part used to make bicontinuous cubic phase contains human plasma, virus-like particles (VLP) which contains the p24 antigen and lysis buffer.
  • VLP virus-like particles
  • the hydrophobic substrate TMB is solubilized in the lipid surfactant Dimodan at a concentration of 1 ,25 mg TMB per 100mg of surfactant, and then mixed with the aqueous part to form the cubic phase.
  • This highly viscous cubic phase ensures the physical immobilization of the antigen p24 with in the mesophase and is then transferred into a glass slide with a neoprene spacer of 0.5 mm, which constitutes a microwell of 0.5 mm in height and 16 mm in diameter. This enables the transfer of fixed amount of mesophase in the glass slide.
  • Mouse monoclonal anti-p24 (Aalto Bio Reagents, BC 1071 , dilution 1 :200) is incubated for 25 min. and then goat anti-mouse IgG conjugated to HRP (KPL, 474-1806, dilution 1 :200) is used as the secondary antibody.
  • HRP horseradish peroxide
  • Introduction of H 2 0 2 on the mesophase promoted the start of enzymatic reaction, yielding a simultaneous color change and birefringence (Fig. 12 a) with in the cubic phase. Absence of birefringence in the POM in 5 different control experiments confirms the specificity of the detection (inset of Fig. 12 a).
  • heat-treated plasma is used to increase the concentration of unbound p24.
  • concentration of unbound p24 antigen is low whereas most of the remaining p24 antigens are bound to the antibody already present in the blood.
  • concentration of unbound antigen can be drastically increased by heating the HIV infected plasma to 100°C for a short duration.
  • the VLP containing cubic phase may be heated to 100° C for 5 min. in a closed vial and cooled to room temperature.
  • the heat-treated plasma in the bicontinuous cubic phase shows a slight background birefringence signal in the POM (Fig. 12 b)
  • the birefringence developed after the ELISA method is significantly higher (inset of Fig. 12 b), which further illustrate the potential of this method in practical application of HIV detection.
  • This birefringence is dependent on the concentration of p24 antigen (Fig. 12 c) and therefore can be used for the quantification of the analyte using the cross-polarized filter device.
  • the measured birefringent intensity from each concentration (I) of p24 antigen and control sample (I0) is calculated and the normalized intensity is used for the quantification (Fig. 12 d).
  • the limit of detection in this case using birefringent ELISA method is 2.5 ng/ml.
  • the detection of analytes using this cheap, portable device by imparting a new optical signal generation mechanism to conventional ELISA is anticipated to be of an unprecedented simplicity for the rapid detection of HIV infection in resource-poor regions. Detection of protozoan parasites
  • the device and method disclosed here can also be used for a label-free, naked-eye detection of Plasmodium falciparum, the devastating infectious pathogen causing malaria.
  • the protozoan Plasmodium parasite invades erythrocytes and digests hemoglobin.
  • the heme component which is toxic to the parasites is crystallized in the form of a brown birefringent crystal.
  • cross-polarized microscopy may be sufficient to diagnose malaria without any additional marker, although this methodology remains difficult to be implemented in the field, especially in malaria-endemic areas, which often are resource-limited.
  • the cubic phase system consists of an industrial grade of monolinolein (Dimodan U/J; Danisco, Denmark), blended with water.
  • HRP Sigma-Aldrich
  • ABTS stock solution of 45.5mM was prepared in pH4.65 (Sigma-Aldrich) acetate buffer.
  • 0.2M H 2 0 2 in pH4.65 acetate buffer was prepared from 50% H 2 0 2 solution in water (Sigma-Aldrich).
  • a two-syringe (Hamilton) coupled system was used for the preparation of the cubic phase at 37°C.
  • the color development was not uniform or started even before the highly viscous cubic phase was formed. Therefore the amount of enzyme and substrate was optimized to get a slow and homogenous color development in the cubic phase.
  • the initial cubic phase was always prepared with a surfactant-to-water ratio of 75:25. This secured a starting Ia3d symmetry; however, for the experiment with bacteria, a Ia3d-Pn3m order-order transition takes place during the rinsing with excess buffer, which however does not affect the birefringence development, nor the main detection mechanism.
  • the cubic phase was prepared in the same way using the OPD stock solution (55mM).
  • TMB Tetramethyl methacrylate
  • HRP H 2 0 2
  • the cubic phase was then prepared as described above. Attempts to reproduce the enzymatic reaction with other substrates such as pyrogallol (Sigma-Aldrich) and o-dianisidine (Sigma-Aldrich) were made but the higher solubility of the converted product did not trigger any crystallization/birefringence.
  • a small amount of the cubic phase was transferred on a clean glass slide, closed with a cover glass and analysed under cross-polarized light using a Zeiss Axioskop 2 mot microscope, at 37°C.
  • Cubic phase samples from the syringe were directly transferred into a demountable UV cell (Starna, Type 20/C/Q/1 ) and UV-vis measurements were carried out on a CARY-100 Bio UV-visible spectrophotometer, at 37°C.
  • the diffracted x-rays signal was collected either by a two-dimensional argon-filled detector (for SAXS) or with the help of a Fuji Film BAS-MS 2025 imaging plate system: 15.2 ⁇ 15.2 cm, 50 pm resolution (for WAXS).
  • the samples were placed inside a Linkam HFS91 hot stage and measured at 37°C. Data were collected and averaged azimuthally to yield one-dimensional intensity versus scattering vector q.
  • ⁇ -D-glucose Sigma-Aldrich
  • ABTS ABTS
  • pH 4.65 buffers The desired concentrations of ⁇ -D-glucose (Sigma-Aldrich) and ABTS (45.5 mM) were prepared separately in pH 4.65 buffers.
  • 100 ⁇ _ of ⁇ -D- glucose and 130 ⁇ _ of ABTS were collected in one syringe while 750 ⁇ _ of monolinolein together with 10 ⁇ _ of GOD and 10 ⁇ _ of HRP were taken in another syringe and mixed through a needle connecting the two syringes as mentioned above.
  • the ⁇ -D-glucose solution was replaced by 100 ⁇ _ of buffer.
  • the required concentration of cholesterol was directly solubilised in the monolinolein.
  • Cholesterol oxidase (ChOD, Sigma-Aldrich) stock solution of 0.2 mg/ml and HRP stock solution of 1 mg/ml were prepared in pH 7 phosphate buffer. 100 ⁇ _ of buffer, 130 pL of ABTS, 10 ⁇ _ of ChOD stock solution and 10 ⁇ _ of HRP stock solution were loaded in one syringe while 750 mg of dimodan together with the required concentration of cholesterol were loaded in the other syringe and mixed as described above. The control sample without cholesterol was prepared in the same way.
  • E.coli (K-12 MG 1655) were cultured overnight on nutrient agar at 37°C. Then single bacterial colonies were selected from LB agar and transferred into 5 ml LB media. After 5 hours, 20 ml additional media were added and incubated overnight at 37°C with shaking at 225 rpm. The following day, cells were pelleted (2500 g, 10 mins) and washed with PBS and pelleted again. Bacteria were suspended as 10 7 ml-1 in pH 4.65 acetate buffer and used within 1 h of preparation.
  • E.coli For the immobilization of E.coli in the cubic phase one syringe was filled with 50 pL of E.coli suspension in acetate buffer (5x10 4 E.coli) and the other syringe with 200 mg of monolinolein together with 2 mg of TMB. Mixing and preparation of the cubic phase was carried out via the double connected syringes as mentioned before. The control samples were prepared without E.coli.
  • E.coli in the cubic phase was stained with 1 pg/ml DAPI (4', 6-diamidino-2-phenylindole) and imaging was carried out with a 100x oil immersion lens (NA 1 .4) with an excitation wavelength of 365 nm and a 420 nm long pass filter for the emission.
  • DAPI 6-diamidino-2-phenylindole
  • An O-ring (1 mm thickness and 8 mm inner diameter) was filled with the mesophase mixed with bacteria and TMB and placed on a glass slide.
  • the sample was washed with blocking buffer (5% BSA solution) followed by rinsing with the PBS buffer.
  • HRP-conjugated primary antibody solution (Abeam ab68450, 1 mg/ml, dilution 1 :100) was added on the mesophase and incubated for 15 min. After washing the mesophase three times with buffer, 0.5 mol/l H 2 0 2 were added to start the peroxidase reaction.
  • a control sample was prepared in the same way without adding E.coli.
  • the P. falciparum 3D7 strain was cultured in vitro in human 0+ erythrocytes at 5 % haematocrit using RPMI medium containing 0.5 % Albumax. Parasites were synchronized with 5 % sorbitol. Cultures were harvested by centrifugation and lysed hypotonically in H 2 0. 100 pL of this lysed culture is then added on top of 50 mg of phytantriol lipid, allowing 3 minutes to form the cubic mesophases in a 96 well plate.
  • Plasmodium falciparum culture (7 % late stage parasitaemia) was pelleted and lysed in 5 ml ddH 2 0 at room temperature for 5 minutes. The lysate was centrifuged at 3500 g for 10 minutes. The pellet was dissolved in 2 % SDS and vortexed vigorously. The lysate was centrifuged for 10 minutes at 21000 g. The resulting hemozoin pellet was twice-washed in ddH 2 0 and stored in 500 ⁇ ddH 2 0 at room temperature.
  • control sample contained 500 ⁇ uninfected human erythrocytes lysed in 1 ,5 ml ddH 2 0.

Abstract

L'invention concerne un procédé pour détecter visuellement une réaction d'un réactif en un produit, et comprend les étapes consistant à obtenir un cristal liquide en mésophase cubique bicontinue comprenant ledit réactif, à obtenir en outre des conditions dans lesquelles le réactif est converti en ce produit dans une étape de réaction, ledit produit étant insoluble dans la mésophase, et à mesurer une valeur de biréfringence de ladite mésophase. L'invention est particulièrement appropriée pour détecter des conversions enzymatiques d'analytes en produits. Par exemple, des conversions en glucose ou cholestérol au moyen de systèmes à oxydase/peroxydase. L'invention concerne en outre un appareil (10) destiné à être utilisé dans un procédé de l'invention, lequel comprend un volume (11) délimité par des éléments optiques transparents (12) et (13) qui sont orthogonaux par rapport à un axe (15) passant à travers ledit volume (11), les axes de polarisation (16) et (17) des éléments optiques pivotant de 90°, et la mésophase cubique bicontinue étant présente dans ledit volume.
PCT/EP2015/062151 2014-06-02 2015-06-01 Biocapteurs à cristaux liquides WO2015185504A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
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