Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
  • G01N29/02—Analysing fluids
  • G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
  • G01N29/02—Analysing fluids
  • G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
  • G01N29/22—Details, e.g. general constructional or apparatus details
  • G01N29/222—Constructional or flow details for analysing fluids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/0877—Flow chambers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2291/00—Indexing codes associated with group G01N29/00
  • G01N2291/02—Indexing codes associated with the analysed material
  • G01N2291/025—Change of phase or condition
  • G01N2291/0255—(Bio)chemical reactions, e.g. on biosensors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2291/00—Indexing codes associated with group G01N29/00
  • G01N2291/02—Indexing codes associated with the analysed material
  • G01N2291/025—Change of phase or condition
  • G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2291/00—Indexing codes associated with group G01N29/00
  • G01N2291/04—Wave modes and trajectories
  • G01N2291/042—Wave modes
  • G01N2291/0426—Bulk waves, e.g. quartz crystal microbalance, torsional waves

Definitions

• the present invention relates to a biosensor cell device and its use in qualitative and quantitative analysis of at least one analyte and/or its interaction partner. Background of the invention
• biosensors consist of two components : a highly specific recognition element and a transducer that converts the molecular recognition event into a quantifiable signal.
• the type of biosensor which is of interest in the present invention is a biosensor operating with a piezoelectric crystal.
• PZ crystals piezoelectric (PZ) crystals as analytical sensors, primarily as microbalances, has been demonstrated in the last years.
• the PZ crystal devices are used to determine the mass adsorbed on the active surface; the material on the surface of the PZ crystals, a monolayer, determines the specificity and selectivity of the sensor.
• QCA Quartz Crystal Analyzers
• the present invention is directed to a novel QCA device or system which is reliable, stable, quick, and sensitive.
• the novel device or system of the invention comprises a closed test chamber cavity which is filled with solvent/water or buffer solution, with or without analyte, and it is particularly suitable for applications where the direction of the test camber may vary, such as in portable detection systems.
• the biosensor cell device of the invention comprises a test chamber cavity and a piezoelectric crystal coated, on the surface facing the test chamber cavity, with an interaction partner to an analyte, the crystal being connected to an oscillation unit and signal processing electronics, which device further comprises an inlet needle for the introduction of the analyte that ends at a small distance from the end of, and inside, an inlet sleeve for the introduction of buffer solution that ends in the vicinity of the active surface of the crystal, the inlet sleeve being connected to an inlet for buffer solution, and an outlet for excessive fluid.
• the inlet needle may be an automatic injector or a syringe.
• the piezoelectric crystal may be coated with two or more different interaction partners each interacting with a different analyte.
• the interaction partner is an antibody which specifically binds to the analyte (antigen), e.g. selected from narcotics and explosives, such as 2,4,6-trinitrotoluene (TNT).
• the interaction partner is a peptide nucleic acid (PNA) oligomer and the analyte is a polynucleotide, or vice versa.
• the polynucleotide may be selected from the group consisting of RNA, DNA and PNA polymers complementary to the PNA oligomer.
• the biosensor cell device according to the invention may be used for both qualitative and quantitative analysis of at least one analyte and/or its interaction partner.
• SHORT DESCRIPTION OF THE DRAWINGS Fig 1. shows a side view of the parts of a biosensor comprising a flow cell/test chamber cavity.
• Fig 2. shows partly a cross-section of the cell where the flow (arrows) is designed to pass over the surface of the crystal for both inlets of buffer and test samples.
• Fig. 3. A : Analysis of 1 ⁇ g of 21-mer oligodeoxynucleotide complementary to 21-mer PNA. B : Analysis of 1 ⁇ g of 21-mer oligodeoxynucleotide with scramble PNA 21-mer.
• Fig. 4 Analysis of 1 ⁇ g of pRK8-hGaIRl with complementary to hGalRl sequence PNA 21-mer.
• Fig. 5 Analysis of total RNA from human melanoma cell line Bowes with PNA 21- mer complementary to hGalRl cDNA. A: 18 ⁇ g of RNA. B: 80 ⁇ g of RNA. Fig. 6. Analysis of dsDNA (348 ⁇ g) from human melanoma cell line Bowes with complementary to hGalRl PNA 21-mer.
• the biosensor cell device of the invention is used in the piezoelectric determination of an analyte, and it comprises a flow cell, in which the solvent/water or buffer solution, with or without analyte, passes over the surface of an oscillating quartz crystal, coated with a layered structure exposing to the solution an interaction partner to an analyte, e.g an antibody complex active against the analyte, such as TNT.
• the interaction partner interacts with the analyte, e.g. antibodies bind to the analyte, in the solution, and the weight difference, at the surface of the crystal, gives a slight change in the resonance frequency in the electronic circuit incorporating the crystal.
• the number of interaction partners e.g. antibodies
• the sensitivity will drop and the cell ceases to function. Then, the quartz crystal is replaced with another one which has a fresh coating.
• this situation will be detected by a signal processing unit, and an automatic or manual change-over to a fresh flow cell takes place.
• the drain pipe from one biosensor flow cell may be directly connected to the inlet of another, thus adding sensitivity to a different analyte, such as an explosive or a narcotic, or a multisensor system of parallel biosensors can be arranged by dividing the inlet into an array of flow cells.
• the bottom part (40) is adapted to the design of the actual crystal (41).
• the crystal is kept in position by the upper part comprising the test chamber cavity (42) , two rubber rings (43) and a sliding outer locking sleeve (44).
• the upper part of the cell is brought into its correct position by a steering pin (46) and two screws (45).
• the cell has an inlet for buffer solution (47), an outlet for excessive fluid (48) and a test sample inlet (49).
• the test sample inlet is shown with a syringe for manual injection.
• the essential details of the biosensor cell device according to the invention are shown in Fig. 1 and Fig. 2.
• the bottom part 40 is adapted to the design of an actual piezoelectric crystal 41.
• the crystal is kept in position by the upper part comprising a test chamber cavity 42 , two rubber rings 43 and a sliding outer locking sleeve 44.
• the upper part of the cell is brought into its correct position by a steering pin 46 and two screws 45.
• the cell has an inlet for buffer solution 47, an outlet for excessive fluid 48 and a test sample inlet 49.
• the test sample inlet is shown with a syringe for manual injection.
• the piezoelectric crystal 41 is coated on the surface facing the test chamber cavity 42 with an interaction partner to an analyte, and the crystal 41 is connected to an oscillation unit and signal processing electronics.
• the inlet needle 49 for the introduction of the analyte ends at a small distance from the end of, and inside, an inlet sleeve 50 for the introduction of buffer solution that ends in the vicinity of the active surface of the crystal 41.
• the inlet sleeve is connected to an inlet 47 for buffer solution.
• the device also comprises an outlet 48 for excessive fluid.
• the inlet needle 49 is an automatic injector or a syringe.
• Examples of different embodiments of the invention are biosensor cell devices wherein the piezoelectric crystal 41 is coated with two or more different interaction partners each interacting with a different analyte; the interaction partner is an antibody which specifically binds to the analyte (antigen); the analyte(s) is (are) selected from narcotics and explosives; the explosive is 2,4,6-trinitrotoluene (TNT); the interaction partner is a peptide nucleic acid (PNA) oligomer and the analyte is a polynucleotide; the interaction partner is a polynucleotide and the analyte is a peptide nucleic acid (PNA) oligomer; and, the polynucleotide is selected from the group consisting of RNA, DNA and PNA polymers complementary to the PNA oligomer.
• the interaction partner is an antibody which specifically binds to the analyte (antigen)
• An other aspect of the biosensor cell device according to the invention is directed to its use for qualitative and quantitative analysis of at least one analyte and/or its interaction partner. Examples of nalvtes
• analytes As the detection targets of the biosensor cell device of the invention two classes of analytes, namely explosives and polynucleotides, have been chosen as preferred embodiments. These two large classes of analytes in turn consist of many different substances. The detection of these with high sensitivity is of enormous importance today, both in the world welfare (explosives) and in biomedical analysis (polynucleotides).
• TNT analysis is achieved by application of antibodies specific for TNT attached to the PZ gold(Au) electrodes.
• RNA/DNA polymers are extracted from human tissue samples and applied to the Cys-PNA-electrode. When the mass increases the frequency of resonance oscillation of the PZ electrode decreases. The results are obtained in form of a diagram where the time is a function of the frequency. The detection of the interaction is obtained in few seconds.
• biosensor QCA method where the interaction between relatively short (18- to 21-mers) peptide nucleic acids, PNA, and any complementary polynucleotides is applied.
• the PNA-PNA, PNA-DNA and PNA-RNA interactions are detected with piezoelectric (PZ) crystals as analytical sensors in the Quartz Crystal Analysis, QCA, system. Trinitrotoluene, TNT, as analyte.
• the antibodies recognizing TNT are used in the Examples of analyses using the biosensor cell device of the invention for the detection of picograms of a small antigen like TNT.
• Polynucleotides as analytes or interaction partners to analytes are used in the Examples of analyses using the biosensor cell device of the invention for the detection of picograms of a small antigen like TNT.
• Detection of polynucleotides as well as their interactions is crucial for biomedical analysis and drug analysis as well as for research.
• the broader goal of the detection of the presence and interactions between polynucleotides with PNA oligomers is to detect various genetically linked disorders. It is well known that many serious disorders such as tuberculosis, malaria, Alzheimer's disease, HIV, cancer, cystic fibrosis etc. are connected to expression of mutated genes or overexpression of certain genes yielding in overexpression of characteristic proteins. In case of occurrence of disorder-linked mutations or overexpression in the case of patients, those will be detected by interaction with complementary to the mutations or overexpressed product PNA oligomers coupled to the PZ crystals.
• PNA Peptide nucleic acids
• the PNA monomers can be polymerized using standard peptide coupling chemistry, which is flexible, highly developed and efficient.
• the hybrids between such PNA oligomers and their complementary single stranded DNA oligomers have shown remarkably high stability (affinity) in comparison with naturally occurring DNA/DNA and RNA/DNA hybrids.
• PNA oligomers offer great advantages in both antisense and antigene approaches for regulating gene expression.
• the PNA oligomers are considerably more stable towards nucleases (compared to natural polynucleotides) due to the lack of (deoxy)ribose backbone.
• Hybridization of PNA oligonucleotides to complementary RNA/DNA sequences by specific base-paring is caused by interaction through hydrogen bonding.
• This simple base-paring allows the design of PNA oligonucleotides that target any gene or RNA of a known sequence.
• a major advantage of this strategy is in the potential specificity of action. High specificity and affinity of the interactions lead to more sensitive detection methods in DNA/RNA estimations.
• the crystal in the biosensor is excited in a modified Pierce oscillator.
• the standard Pierce oscillator is modified with: - an extra buffer stage in the oscillating loop to compensate for the high damping of the crystal by the fluid,
• the signal processing electronics contains the electronics for driving the crystal, measurement of different parameters and decision algorithms for evaluation of the results.
• the electronics for driving the crystal and measure the frequency shift consists of an oscillator (Colpitt or Pierce style) with the crystal followed by a buffer amplifier and a fast precision counter with the interpolation.
• the results from the frequency measurements are fed to the control computer for evaluation.
• the electrodes used in the Examples are AT-cut 9 - 10 MHz piezoelectric crystals with gold electrodes, supplied by Quartz Probe, Sweden, or Seiko, Japan.
• B2 0.01 M Na-phosphate buffer, 0.1 M NaCl, pH 7.4, consists of Na 2 HPO 4 .12H 2 O: 3.58 g/1; NaH 2 P0 4 .H 2 O : 1.38 g/1 ; NaCl 5.85 g/1. Adjust pH with the salts
• Water Deionized tap water Thiol or disulphide containing modification reagents can be used with Au-electrode TNT analysis
• TNT-antibodies were used. TNT-antibodies are commercially available. Polynucleotide analysis
• Peptide synthesis reagents described in Design of chimeric peptide ligands to galanin receptors and substance P receptors. Int. J. Pept. Protein Res. 39, 516-522. (1992) Langel, Ulo., Land, T., and Bartfai, T.
• the ethanol-washed and dry electrode was incubated in the 1 ⁇ M solution of the appropriate Cys-PNA 21-mer solution in B2 for 14-16 h at room temperature in dark and in tightly closed tube. The electrode was washed with water, incubated for 5 min in water. The PNA-modified electrode was placed into the cell and washed with B2, 100 ml/h, 10 ml, the electrode was stabilized (change is less than 2 Hz/10 min) for 20 - 60 min.
• 21-mer cPNA complementary to region 18-38 of the human galanin receptor type 1: GC GTT GCC CTC GCT GAG GTT C amide 21 -mer scrambled PNA: GGC ATG GCT GCT CTC CGT CTG amide
• aqueous (top) layer was transferred to a new tube and 1/2 vol of 7.5 M ammonium acetate and 2 vol of 100% ethanol were added.
• the pellet was rinsed with 75% ethanol, ethanol was decanted and the pellet was dried. DNA was resuspended in TE buffer until dissolved.
• RNA pellets were lysed by adding UltraspecTM RNA. 0.2 ml of chloroform per 1 ml of UltraspecTM RNA was added and shaken at 4°C for 5 min. The homogenate was centrifuged at 12 000 x g (4°C) for 15 min. The aqueous phase was carefully transferred and equal volume of isopropanol was added; the samples were incubated for 10 min at 4°C. The supernatant was removed and the RNA pellet was washed twice with 75% ethanol by vortexing and subsequently centrifuged for 5 min at 7 500 x g at 4°C. The pellet was dried under vacuum for 5-10 min. The RNA pellet was dissolved in 50-100 ⁇ l UltraspecTM DEPC treated water.
• Regeneration of the electrode is achieved by adding RNAses or DNAses which degrade the specifically bound RNA or DNA, respectively.
• Another approach to prepare the once used PNA-electrode for the next application is to wash the specifically bound polynucleotides by washing of the cell with the buffers with different ionic strength and water. 10.
• the equilibrium of binding of oligonucleotides to PNA-electrodes is achieved within 100 - 200 s, the time which is convenient for determination of the interaction kinetics without additional equipment. Hence, the method is applicable for measurement of the kinetics of the interaction between the PNA and analyzed RNA or DNA monomers or dimers. 11. Immobilization of biotinyl-PNA polymer to streptavidin coated electrode
• biotinyl moiety into the appropriate sequence of the PNA polymer. It is possible as well to coat the QCA electrode with avidin or streptavidin, and to use the coated electrode to bind the biotinyl-PNA. This method is alternative to the coupling of PNA polymers to the electrodes by reaction of SH-groups with gold surface (cf. above).
• Rat Rin m5F cells were cultivated in RPMI 1640 (Gibco 041-01870), supplement with 5% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin. The cells were grown over 3 days to about 50 % confluence in tissue culture flasks.
• Rat SHSY cells were cultivated in Minimal Essential Medium with Earles salts (MEM), supplement with 10 % fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 ⁇ g/ml streptomycin, 1 % non essential amino acids. The cells were grown over 3 days to about 50% confluence in tissue culture flasks.
• MEM Minimal Essential Medium with Earles salts
• PCT EP98/03531 the contents of which is incorporated herein by reference, in detection of the small amounts of TNT in air probes collected from ground presumably containing mines followed by concentration.
• the construction of the biosensor cell of the invention permits the stabilization of the system in 1-2 min or faster.
• the method of TNT detection by application of specific antibodies on QCA resulted in detection of TNT at the low level of 10 pg TNT.
• B. Polynucleotide analysis The initial analogs of PNAs belong to the complementary human and rat brain galanin receptor cDNA-derived sequences (cf. above). The 21-mers from human galanin receptor cDNA serve as lead sequences due to their demonstrated high specificity in preliminary experiments.
• Electrodes immobilized with rat and human PNA 21-mers were exposed to RNA from cell lines not expressing the galanin receptors, SHSY.
• DNA deoxyribonucleic acid
• Notl a restriction endonuclease from Nocardia otitidis-cavarium
• PNA peptide nucleic acid pRK8: mammalian vector derived from vector pRK5
• RNA ribonucleic acid
• UltraspecTM DNA and RNA isolation reagent, contents : phenol, guanidine salts, urea, buffering agents, detergent and stabilizer.

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Citations (4)

• 1998
  • 1998-09-24 SE SE9803238A patent/SE512994C2/sv not_active IP Right Cessation
• 1999
  • 1999-09-22 WO PCT/SE1999/001658 patent/WO2000016903A1/en active Application Filing
  • 1999-09-22 AU AU64916/99A patent/AU6491699A/en not_active Abandoned

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