TWI252921B - Bio-vehicle, biosensor and biotransducer system - Google Patents

Abstract

A bio-electronic system for detecting a broad range of chemicals is achieved by the incorporation of ligand-binding affinities of lipocalins with a mass-sensing electronic device. A biotransducer system combines the unique ligand-binding affinities of lipocalins with the high sensitivity of piezoelectric-based devices. As ""artificial-nose"", this system is more reliable, cost-effective, and flexible for a number of applications in sensory evaluation, including air quality monitoring, quality control of cosmetic and fermentation products, medical diagnosis via breath analysis et al. These proteinaceous elements are likely to be extended to several potential applications, e.g., for the purification and delivery of specific ligands.

Description

1252921 V. INSTRUCTIONS (1) 1. The technology of the invention belongs to i or the case relates to a kind of biological carrier, especially if it is taken and retained as odorous substances, 仏4, gentleman, moxibustion white matter, and can be caught. The present invention also relates to a bio-color 3 low-molecular-weight chemical biological carrier, and can detect a chemical substance "a bio-containing protein-related bio-transducer system, especially a combination of a carrier, and is detected. The chemical species contains protein-like bio-electronic signals. The combination of the present invention and the biological carrier produces 4^ Ming also relates to the use of the above device. Oral presentation 2, prior art item $ artificial 的 的 市 是 是Phase / ^ > system, the array of chemical sensing workers; = mixed and expensive. The transmission is 'this way has some shortcomings: Department: such as the high selectivity of the detectors (ligions) In fact, the detection specificity of the current system is only rare and difficult to find. Another: Bu, its output must be processed by hardware such as pc = sample ligand. When a variety of analytes are used, their output is often very complicated. Objective: When applying chemical sensing technology, the "manual olfactory" system is used, and the information obtained is used, because of &, in the current platform. At a high price, the so-called The biological system that simulates various biological sensors is designed to design the first biological or raw (four) raw sensing device. The generation of biosensors to meet more needs: ^ type or factory online monitoring, has been used commercially : 'The home sensor is used as an important standard. Therefore, the current or = glucose _ π can not be expected can be page 9 1252921 5, the invention description (2) portable instrument can be used by the user using the sensing element test piece ( The replacement of ch ip ) to design the biosensors required by the company. This device was first seen in the "HAZMATCAD" product manufactured by Microsensor Microsensor Systems, Inc., USA, and Cyranose 3 2 from Cyrano Scientific, Canada. 0, 'In the product, and published by the Sandia National Laboratory in the United States, Chem Lab on a Ch lp (Chemical Laboratory on Test Strips). Despite this, the extended application of these artificial olfaction is still limited by the type of sensing element, which is difficult to manufacture on a large scale and sold at a low price. In biological systems, odor recognition occurs first when odor molecules bind to specific protein receptors in certain organs or tissues. In one method, biological materials are utilized as part of the electronic nose of various sensing technologies, and therefore, the selection of biological materials is diverse. Preferred biological materials can be purified olfactory receptors (e.g., Wu, Biosens Bioelectron. 14:9 (1 999)), antibodies (e.g., wong et al., Bi〇sens.

Bioelectron·17··9 (2 0 0 2), fish scales (as revealed by Lundstr〇m and Svensson, Biosens. Bioelectron. 13:689 (1998)), insect tentacles (eg Schroth et al, Electrochimica)

Acta.44: 382 1 (1 999), and synthetic peptides (eg, Wu and Lo, J. Biotech 80:63 (2000). However, these systems are cost and practical. Other factors, it seems difficult to use in industrial production. Therefore, it is necessary to find the ideal biological material for a wide range of sensing applications.

Page 10 1252921 V. INSTRUCTION DESCRIPTION (3) The present invention provides a biological carrier which is made by potting a chemical substance such as an odorous substance, a drug or a pigment. At the end of the capture, a biodetector is additionally provided, which is suitable for sale at a low price. The human rule can be used to make a wean and the invention can additionally provide a test application. "The system is suitable for a variety of senses. The first aspect of the invention relates to an egg carrier, a biological carrier for binding to a phase-matching ligand, which includes: - solid energy * private use ^ back to the sorrow support; and a protein body, which is housed by the solid support and can be combined with the specific ligand.

The protein matrix comprises a hydrophobic molecule binding protein family (Hp〇calin protein family) and its recombinant functional homo 1ogues, which can be a disposable biological test piece (bio) -chip) or a reusable container. The biological carrier of the present invention is particularly suitable for combination with a specific ligand having a molecular weight of less than 1 〇〇〇 吞 (D a 11 ο s ). Examples of the specific ligand include odorous substances, drugs, and pigments. For example, 2-phenylethyl alcohol (2-

Phenylethyl alcohol, 2-phenylethyl acetate, phenylacetate, propylbutyrate, hexylbutyrate, benzylbutyrate , propyltiglate, hexyltiglate, and benzyltiglate. An example of this protein matrix is the major urinary protein (major urinary)

Page 11 1252921 V. INSTRUCTIONS (4) protein (MUP)) The second aspect of the invention relates to a biodetector for detecting the binding of a specific ligand, including a piezoelectric group. a material that generates a charge in response to a mechanical stress; and a protein matrix immobilized on the piezoelectric substrate to provide the mechanical stress to the piezoelectric substrate when combined with the specific ligand. A third aspect of the invention relates to a biotransducer system comprising: a protein matrix for binding to a specific ligand; and an electronic detection device coupled to the protein matrix for detecting the specific The ligand binds to the protein matrix and produces an electronic signal to represent the presence of the particular ligand. Preferably, the protein matrix comprises a protein selected from the group of hydrophobic molecular binding proteins and its recombinant functional homologs. The electronic detector preferably includes a piezoelectric based device and a recorder. The piezoelectric based device may, for example, be a quartz crystal microbalance (QCM) or a surface acoustic wave (SAW) element.

A fourth aspect of the invention relates to a method for detecting a combination of a specific ligand and a protein matrix, comprising the steps of: providing an electro-substrate; and fixing an f-white matrix to the piezoelectric substrate; The protein matrix and the piezoelectric substrate are exposed in the presence of the specific ligand, and the oscillation frequency of the piezoelectric substrate is measured to determine that the specific coordination has been bound to the protein matrix. A fifth aspect of the invention relates to the use of the biological carrier for sucking

1252921 —-—--- V. INSTRUCTIONS (5) Attach, store, purify or deliver the specific ligand. For the month, the sixth aspect relates to the use of the biotransducer system, articles; two = 1, air quality monitoring, cosmetic quality control, fermentation, or medical diagnosis by respiratory analysis. . BRIEF DESCRIPTION OF THE DRAWINGS The present invention can be better understood by referring to the following drawings, in which: Figure 1 is a schematic block diagram of a main portion of a transducer system;

The second figure is a diagram showing the frequency versus time of the electronic signal emitted by the bio-transducer system in a preferred embodiment of the present invention, wherein air is introduced into the system, and the blank test is performed first; In a preferred embodiment, the frequency versus time relationship of the electrical signals output by the bio-transducer system as shown in the first figure, wherein a two-times ligand sample is introduced into the system for bonding test; In the preferred embodiment of the present invention, the frequency versus time relationship of the electrical signals outputted by the bioswitch system as shown in the first figure, wherein a low concentration ligand sample is introduced into the system for bonding test;

Figure 5 is a diagram showing the frequency versus time of an electrical signal outputted by a biotransducer system as shown in the first embodiment in a preferred embodiment of the present invention, wherein high and low concentration ligand samples are introduced into the system. Performing a binding test; Figure 6 is a frequency versus time diagram of an example of a biotransducer system of the present invention, showing the combination of volatile odorants produced by the yeast culture at the beginning of the fermentation; and the seventh figure is Frequency and time of an example of a biotransducer system in this case

1252921 V. INSTRUCTIONS (6) -------________ Relationship diagram, using the volatility of lifetime ^ to show the combination of the yeast culture used in the sixth plot in the fermentation t period. Schematic component number Protein matrix 2 0 Piezoelectric substrate 31 Ligand chemistry 1 0 Electron detection device 3 记录 Recorder 3 2 MODE The invention will be described in more detail by way of the following examples. The following preferred examples are for illustrative purposes only, and the present invention should not be limited to the details. Referring to the first 'system 1', the body chemical 1 〇 device 30, with the mechanical stress of the detecting device 30 And the electric substrate 31. The single portable device protein matrix 20 matrix 20 combines to generate a charge, and a sensory evaluation is achieved. A biotransducer system, including a white matter component, can be different from the biotransducer system. The generation of different electro-electric substrates (piezoelectric charge, and a recorder 32, in order to provide a routine sense is not limited thereto. The piezoelectric substrate 31 is applied to the piezoelectric device when mechanical stress is applied. Record the signal. Gas quality monitoring, cosmetic products or multiple sets of eggs. The ligand contains a wide range of pressure proteins containing an electrical signal. base) Electrical connection measurement of the volume and substrate 31 Quality control page 14 1252921

The biotransducer system preferably combines with a ligand chemical such as an odorant, drug or pigment, for applications such as quality control of fermentation products, or medical diagnosis by respiratory analysis. One or more proteins contained in the protein matrix 20 are preferably selected from the group of hydrophobic molecular binding proteins (lip0calin pr〇tein

Recombinant functional homo 1 ogues. An in-depth discussion of hydrophobic molecular binding proteins can be found in Biochimica et Biophysica Acta (BBA) ~ Protein Structure and Molecular Enzymology 1 482 (2000). The hydrophobic molecular binding protein system is a rich protein group (more than 140 species) found in eubacteria and various eukaryotic cells. Its main biological functions are pheromonal carriers, vitamin A transmitters, olfactory and taste proteins, pigments, prostaglandin D synthesis, immunomodulation, stress proteins, and Regulation of cell differentiation. Although the overall sequence similarity is low (usually less than 20%),

However, members of the hydrophobic molecular binding protein family share a common feature, namely eight-stranded anti-parallel barrels with a repeating +1 phase surrounding an internal ligand-bound bag (eight-stranded antiparallel - barrel with a repeated + 1 topology enclosing an internal ligand-b indingpocke ΐ ), detailed description of the features can be found at www.jenner.ac.uk/lipocalin.htm 〇 Most of the hydrophobic molecular binding proteins are abundant and can be naturally

Page 15 1252921 V. Description of the invention (8) The main protein contained in the source, for example, is the hydrophobic protein-binding protein/5-lactoglobulin (BLG). Recombinant forms are also known and available in the art, such as retinal-binding proteins, see Wang et al.,

Gene 133(2): 2 9 1 (1 9 93 ), tear lipocalin, see Holzfeind et al., FEBS Lett. 395(2 - 3): 95 (1996) , cattle / 3 - lactoglobulin, see Cho et al,, Protein Engineering 7 (2): 263 (1994), major urinary protein, see Ferrari et al, FEBS Lett· 40 1 ( 1 ): 73 ( 1 99 7 ), Nitric Oxide Delivery Proteins, see Andersen et al., Structure 6 (1 0 ): 1 3 1 5 (1 9 98 ), and odor molecules For binding proteins, see L〇bel et al., European J. Biochem 254(2): 318 (1998) One example of a hydrophobic molecule binding protein is the major urinary protein (MUP). MUP is the main protein component in the urine of mice (male about 5-10 love per day: gram). Can be used as a pheromone carrier, see cavaggi〇ni and

Mucigna, Caretta, BBA 1482 (2-1): 218 (2000). Reorganization

MUP can be made by standard procedures known to those skilled in the art. MUP is particularly good for carrying low molecular weight, high volatility compounds below 1000 Torr. One embodiment of the piezoelectric substrate 31 is a quartz crystal microbalance (QCM). The preferred electronic olfactory technique of the present invention is based on piezoelectric technology. By detecting an analyte (ligand) and immobilizing it on a piezoelectric material (such as a quartz crystal)

Page 16 1252921

The combination of sensing elements is used for measurement. The adsorption of the ligand induces a shift in the oscillation frequency' whose offset is proportional to the mass of the associated ligand. The mode of operation of the present invention is as follows: i) Surface activation of the QCM test piece The quartz crystal microbalance (QCM) surface is immersed in 1. 2 N NaOH, and then cleaned in 1 · 2 N H C 1 . After infiltration and drying, the q c μ electrode was immersed in a 1 mM 2-aminoethanethiol/DMSO solution for 12 hours and washed with double steamed crane water for 10 minutes. Next, the QCM electrode was dried in an oven and dropped with 4 μl of 2.5% pentose < glutaldehyde for 1 hour. Two microliters of a 1% solution of 1% polyethyleneimine/methanol (pol yet hylen imine/met hano 1 (PEI/MeOH)) was added to the qcm activated surface at room temperature in a sealed chamber for 12 hours. Finally, the QCM test piece was washed twice and dried in an oven. i i ) Fixing the MUP on the surface of the activated QCM test piece

The QCM test piece activated with 25% 5% glutaldehyde was added for 1 hour. Then, the MUP solution was continuously added for 1 hour (20 μg/ml in 1 mM mM sulphate buffer, pH 7.4). . The qcm activated surface was separated by 100 mM glycine. i i i ) Determination of ligand binding The samples were placed in a syringe and an oven at 50 ° C. The adsorption of the odor gas was measured using an AT-cut QCM apparatus having a fundamental oscillation frequency of 10 MHz. The evaporated odor sample was aspirated into the QCM detection chamber and the resulting frequency offset was recorded. Figures 2 through 5 depict the various outputs of the same biotransducer system

Page 17 1252921 V. INSTRUCTIONS (10) The frequency versus time of the sample. Rate ί Combine the test sample with air. Because the flow of air causes some kind of private power, the frequency of vibration of QCM increases. "No.: The test samples combined with the four figures are 40 // 1 and 4 // Christine 7 mt ^ ). No, the ligand adsorption induces: 'two, {shift, its offset and The quality of the relevant ligand is proportional. It also shows that the system of the present invention can also detect the diluted sample. The ligand binding test sample of the fifth figure is sequentially 4 "1 phenyl acetate, ::, and 40. // 1 | acetate. It can be seen that the adsorption and desorption of the sample is good. Thus, the system of the present invention can be reused and has the potential for quantitative analysis and on-line assays. For biochemical gas testing, the present invention analyzes a septic sample relative to a yeast culture exposed to 丨〇 cM. The pichia ferment ans L-5 was cultured in a 4 liter fermentation tank having a working volume of 丨·6 liters, a gas volume of 0.6 vvm (air volume per minute/tank volume), and a rotation speed of 4 Torr. The initial medium contained phenylalanine 2000 ppm, glucose 1 〇〇 g/L, and 5 mM KHJO4. The odor substances expected in such culture exposures include 2-phenyl ethyl alcohol and 2-phenylethyl acetate. The sixth and seventh figures show the combination of odorant and protein# matrix in the early and middle stages, respectively. The invention is clearly visible on both quantitative and on-line measurements. The potential of 疋 According to the above steps to study a variety of ligands. Table I lists with different coordination

Page 18 1252921 V. INSTRUCTIONS (11) Signal changes during bone recombination, including phenylacetate, propylbutyrate, liexylbutyrate, house butyrate (benzylbutyrate), propyltiglate, hexyltiglate, benzyltiglate.

Table I Ligand phenylacetate butyl butyrate butyrate butyrate signal meal (frequency / representation) 46 26 23 43 Ligand propamate crotonate crotonate signal change (frequency / indicated) 29 55 43

In addition to the QCM, the electronic detection device used in the system of the present invention may be a field effect transistor (FET) device or a surface acoustic wave (SAW) device. The principles and operation of such devices 4 are well known to those skilled in the art and will not be described herein.

In addition to being used in biodetectors or biotransducer systems, the protein matrix can also be used as a biological carrier for adsorption, storage, purification, and differential use of specific ligands. The protein-based system is immobilized on or contained in a solid support, which may be a disposable orchid test piece or a reusable container. The present invention has been described in terms of the most practical and preferred forms at present.

1252921

Page 20 1252921 The diagram briefly illustrates the second output of the electrical system in which the third object transducer is introduced into the Nanneng fourth object transducer to introduce the low concentration fifth object transducer to introduce the high and sixth relationship diagrams, the odorant The seventh diagram, the first vapor of the transducer is a schematic block diagram of the main part of a transducer system; in a preferred embodiment, the frequency versus time of the biotransducer system, wherein the air is introduced into the map The blank test chart of the sub-signal of the case is the ligand map of the system. The system is the system of the system. The low-concentration map of the system is used for the case. Preferably, the telecommunications sample of the preferred odorant is selected from the preferred telecommunication sample to the telecommunication sample to optimize the biotransformation of the telecom sample in the initial state of fermentation; In the frequency system of the embodiment, the medium frequency of the embodiment is carried out in the frequency system of the embodiment medium to the period of the system middle system, and the yeast and the system are used for yeast cultivation. situation. , such as the first and the time diagram combined test, such as the first and the time diagram combined test, such as the first and the time chart shown in the test; the map is tested; the relationship diagram is combined with the test of an example of the frequency culture produced Health, in which the life and time of the volatilization of the example shown in the figure, the frequency and time of the nutrient produced in the middle of the fermentation

Page 21

Claims (1)

1252921 VI. Patent Application 1 • A biological carrier for binding to a specific ligand, comprising: a solid support; and a protein matrix, which is housed by the solid support and can be associated with the specific ligand The protein matrix comprises a hydrophobic protein binding protein family (lipocalin protein family) and a recombinant functional homologue thereof (Recombinant functional homo 1 </ br> </ RTI> 2, wherein the solid support is one A bio-chip according to claim 1, wherein the solid support is a reusable container. 4 · The biological carrier of claim 1 Wherein the specific ligand has a molecular weight of less than 1000 Daltons. 5. The biological carrier of claim 1, wherein the specific coordination system is selected from the group consisting of odorous substances, drugs or pigments. The biological carrier of claim 1, wherein the specific coordination system is selected from the group consisting of 2-phenylethyl alcohol, B 2-phenylethyl acetate, phenylacetate, propylbutyrate, hexylbutyrate, benzylbutyrate, crotonic acid Propyltiglate, hexyltiglate, and benzyltiglate 7. The biological carrier of claim 1, wherein the protein matrix comprises major urinary protein (MUP) )). Page 22