JP3226373B2 - Biomolecule switch - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to the field of information technology, and more particularly to the bioelectronics and construction of biomolecular switches. The biomolecule switch according to the present invention uses a biopolymer such as protein, nucleic acid or polysaccharide, and is used for a data collection and processing device or a biosensor.

[0002]

2. Description of the Related Art Digital computers typically represent variables in a quantized or digital form. Thus, the individual values of the analog variables are represented using one of many discrete states or conditions. For example, 1
The 0-ary system uses digits from 0 to 9. However, 10
Systems are extremely rare because they tend to require complex arrangements of components during manufacture and are consequently expensive. This is true even for systems with more than two states. Therefore,
A binary system using only two states represented by only 0 and 1 is commonly used. An example of a simple system with two states is a mechanical switch (on / off) and a transistor (conduction / shutoff). Such systems are typically built on tiny solid-state circuits consisting of interconnecting semiconductor elements printed on silicon chips. However, with the rapid growth of information technology, the demands for higher integration and the manufacture of smaller information processing devices continue to increase. While the new developments in precision printing technology have allowed the manufacture of smaller devices, the physical constraints (e.g., heat loss) of commonly used materials, typically silicon and gallium arsenide, have imposed these devices. To improve the integration and miniaturization of the device beyond a certain level.
As a result of the desire for further miniaturization and higher integration of structures, the use of organic materials in the field of molecular electronics, especially the possibility of using biopolymers for such purposes, has been discussed. ing. For example, Conr
ad (M) Common ACT 28 464 (1985) describes a use of the enzyme in biomolecular computer. The computer takes advantage of the ability of the enzyme to read the concentration gradient formed by consumption of the substrate by the enzyme.

[0003] Biopolymers have the advantage of being relatively inexpensive, having high thermodynamic efficiency, and exhibiting self-assembling capabilities. Thus, some of the synthetic problems associated with the use of organic and inorganic molecules are avoided.

[0004] Such polymer-based devices have the potential to achieve extremely high recording densities and are useful for the development of non-Von Neumann-type structures that were previously impossible with digital computation.

[0005]

SUMMARY OF THE INVENTION The present invention exploits the ability of biopolymers to change physical / chemical properties in response to specific stimuli, or in response to perturbations in their microenvironment, and to utilize this property. Is intended for use in the production of biomolecular switches based on the different states of these macromolecules.

Accordingly, the present invention provides a biomolecular switch suitable for use in biosensors and data collection / processing devices. This biomolecule switch includes a sequence of a biopolymer immobilized on a carrier, which can have two or more kinds of existing states that are stable in identification degree. Because the interconversions between these states are rapid and reversible and are induced by the application or removal of an appropriate external stimulus, this macromolecule selects between the unstimulated state and the stimulus-dependent state. It is possible to perform mutual conversion. In order to take advantage of this property of the polymer, it is necessary to first apply a stimulus selectively to the macroscopic environment of the polymer, and to stimulate at least a part of the polymer from the state without stimulation. It can be converted to a dependent state (input), and secondly, it can measure or monitor changes in the state of the macromolecule (output). The present invention
Suitable for use in sensors or data acquisition and processing equipment
A biomolecule switch, comprising:
An array of immobilized biopolymers, wherein each polymer is:
Two or more reversible interconversions
A living organism capable of existing in a state and interacting in the sequence
Sequence of macromolecules; at least some of the macromolecules
The macromolecule to convert from a state to a stimulus-dependent state
An input means capable of selectively applying a stimulus; and the macromolecule
Output means to measure or monitor changes in
A biomolecule switch comprising:

The conversion of input to output is regulated by the polymer described above and depends on the properties of the particular polymer utilized. The selected polymer can be a natural polymer, a synthetic polymer, a chemically modified polymer, or a polymer obtained by genetic engineering techniques.

[0008] As a means for the input means, pH or temperature or ionic strength Doma <br/> other in microscopic environments polymer electrically or with regulatory function of the ligand concentration or any combination thereof Electromagnetic means are preferred. As a means for output, a means adapted to measure the relative quantity of the state at the time of non-stimulation and the state dependent on stimulus is desirable.

[0009]

DETAILED DESCRIPTION OF THE INVENTION Biological macromolecules such as proteins, nucleic acids and polysaccharides, especially proteins, can exist in more than one state with comparable stability. These states represent differences in the three-dimensional structure, chemical or physical properties, quantum mechanical electron spin states, and / or biological activity of the polymer.

[0010] Proteins are particularly useful for the purpose of constructing such biomolecular switches.

The reason for this is the sensitivity of the protein to changes in the microscopic environment, for example, changes in pH, temperature, ionic strength and ligand concentration can affect the physical and chemical properties of the protein. These changes in the microscopic environment are generally expressed as changes in the structure of proteins,
Often accompanied by a change in the biological activity of the protein. Such structural changes typically occur in millisecond time units.

It is important to note that extreme pH, temperature or ionic strength can cause irreversible changes in the state of the protein, and to what extent the protein can tolerate perturbations in its microscopic environment. It is clear that it depends. For example, many proteins are irreversibly denatured at temperatures between 40 and 50 ° C. However, other proteins exhibit higher thermal stability and are able to withstand high temperatures, and such proteins are particularly useful in the construction of biomolecular switches.

The term “microscopic environment” in the present specification
Indicates the local environment of the polymer. As used herein, the term “ligand” refers to any molecule or ion that can reversibly bind to a macromolecule. The ligand to be selected will depend on the macromolecule chosen, but examples of suitable ligands for use in proteins include substrates, antagonistic and non-antagonistic reversible inhibitors, negative and positive effectors, and There is a factor.

[0014] The affinity of a protein for any particular ligand may be further regulated by external factors such as the pH, ionic strength, and temperature of the surrounding microenvironment. For example, a change in pH can affect the ionization of certain acidic and basic amino acid side chains that are essential for the binding of its ligand. In addition, the binding itself may also alter the chemical or physical properties of the ligand.

[0015] Nucleic acids can also be used in biomolecular switches. Extreme pH, temperature or ionic strength results in the reversible dissociation of double-stranded DNA, which is characterized by a decrease in viscosity (this phenomenon is called melting). When the pH, temperature, or ionic strength is returned to the normal physiological range, the dissociated duplex will spontaneously anneal (ie, rewind) to form the original double helix structure.

Examples of macromolecules utilized in the present invention include polysaccharides, nucleic acids, and proteins such as receptors, enzymes and glycoproteins. Preferably, the macromolecules used to make the biomolecular switch should be pure, but some impurities can be tolerated. The biopolymer is preferably available in a large amount and is thermally stable. Particularly useful in this regard are polymers that maintain their structure at temperatures above 50 ° C.

To date, macromolecules that have been the subject of much research and have provided detailed information on their structure,
Particularly useful. This is because such information can easily be used to determine what chemical modification is beneficial and to select an appropriate technique for immobilization. Proteins useful in the context of bioelectronics are enzymes and receptor proteins involved in cellular information transduction systems.

Any appropriate carrier can be used for the biomolecular switch of the present invention. For example, metal, glass, or synthetic polymers can be used as a carrier. However, when the input is a change in ligand concentration and the output is determined by the number of bound ligands, the polymer is immobilized on the carrier by a method that does not use the functional groups necessary for binding the ligand. Is important. Various immobilization techniques have been developed by LQZhou and AEGCas
sensors Bioelec. 6 445-450 (1991).

The carrier is preferably quartz. Quartz has the following advantages. (1) Silanization of quartz with silanes having organic functional groups provides various routes for immobilizing macromolecules, especially proteins. (2) A metal film can be easily formed on quartz before immobilizing the selected polymer, and this film can be used as an electrode for controlling the environment of the polymer. (3) When a polymer having intrinsic fluorescence or a fluorescently labeled polymer, particularly a protein, is used and excited, quartz absorbs much less light on the blue wavelength side than glass and synthetic polymers. Therefore, fluorescence emission can be detected (collected) efficiently, and light emitted from the surface of the quartz carrier can be guided as an evanescent wave, so that light can be efficiently collected.

A second preferred carrier is aminopropyl glass beads, where the porous structure provides a large surface area and the active or amino groups are present. Glutaraldehyde, carbodiimide and triazine are particularly useful reagents for crosslinking between the polymer and the carrier.

The nature of the input is, of course, determined by the type of polymer selected. However, generally electrical input is used, either pH or temperature Doma <br/> other in microscopic environments or resulting in a change in the ionic strength or any combination thereof, or introducing a ligand, or certain Or effect any combination of these . The biomolecule switch of the present invention may include a means for applying an electric potential on the immobilized polymer sequence, if necessary. The potential difference imparted on an array of macromolecules changes the microenvironment of individual macromolecules by adjusting pH or temperature or ionic strength or ligand concentration or any combination thereof , so that:
It can be used to alter the structure of the macromolecule or the frequency at which ligand binding occurs. Biomolecular switches that require the use of voltage for operation should obviously have electrodes for applying current.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In an embodiment of the present invention, a biomolecular switch includes at least one electrode coated with a conductive polymer. This electrode can be used to selectively release or remove ligands (anions or cations) relative to the macromolecular array. This release and removal depends on the applied voltage. In addition, the conductive polymer can change the p ion concentration by changing the hydrogen ion concentration [H ⁺ ].
Can be used to adjust H. The amount of ligand released or the degree of pH adjustment will obviously depend on the length of time the voltage is applied and the value of the voltage within a particular voltage range. If the polarity is reversed, the effect is also reversed.

In an embodiment of the present invention, increasing the potential has the effect of forming a concentration gradient of ligand or pH on the macromolecular sequence. Such a biomolecular switch is particularly useful as an algorithm or a memory for conversion. In these systems, the applied voltage (input) is used to control the macroscopic environment of the polymer,
The output is a response pattern from the arrangement of macromolecules adjusted by the change in the stimulation pattern.

When the ligand concentration is adjusted as described above, the ligand concentration is not only dependent on the voltage but also on whether the existing state of the individual macromolecules is ligand-bound or non-ligand-bound. Dependent. That is, the state of ligand binding of any one macromolecule depends on the state of ligand binding of surrounding molecules. Thus, for any given voltage applied for any amount of time, there are different profiles of ligand-bound and non-ligand-bound macromolecules.

The change from one state to the other can be accomplished by monitoring changes in the intrinsic properties of the macromolecule and / or ligand, or by adding a detectable label to the macromolecule or ligand. Can be tracked. Any label that is easily detectable can be used. The choice of label depends primarily on the macromolecule chosen for the production of the biomolecular switch. Methods for labeling macromolecules and ligands are known and include, for example, M. Brinkley
Bioconjugate Chemistry 3 2-13 (1992) describes the range of available techniques.

Examples of methods that can be used to monitor changes in state include nuclear magnetic resonance, infrared spectroscopy, UV difference spectroscopy, fluorescence, chemiluminescence and bioluminescence.

According to one aspect of the invention, a biomolecular switch is used in a biosensor and acts as an input sensor. The output, ie the relative quantity of the two states of the macromolecule, is a direct function of the amount (input) of the applied stimulus. According to yet another aspect of the present invention, the macromolecule is used as a sensor of an auxiliary input and can simultaneously convert two inputs into a single output. The output here is a function of the effect of the auxiliary input on the original input-output response.

An embodiment of the present invention will be described below with reference to the drawings.

Example 1 Riboflavin Binding Protein Riboflavin binding protein (RBP)
At pH values above 4.5 it binds strongly to riboflavin (ligand) (Kd = 10 ⁻⁹ M), but at pH values below pH 4.0 it readily releases riboflavin. As shown in FIG. 1, the intrinsic fluorescence of riboflavin disappears upon binding to RBP, and this fluorescence is regained only by the release of riboflavin.

The pH of the macroscopic environment of the polymer is controlled electrochemically by changing the potential. Thus, the binding of riboflavin to RBP is regulated by electrical input, and the switching between the two states of the resulting protein (ligand-bound and non-ligand-bound) is followed by monitoring changes in fluorescence. Is done. In order to control the binding of riboflavin to RBP, it is first necessary to know the binding activity of the RBP used and the effect of pH on the binding reaction.

(A) RBP Binding Activity A constant riboflavin concentration (0.5 μg) was titrated with RBP. FIG. 2 shows that about 60 μg of RBP was required to quench the fluorescence of 0.5 μg of riboflavin, and that its molar ratio (RBP / riboflavin) was 1.3. RBP is known to bind to riboflavin in a 1: 1 ratio. The slightly higher molar ratios described above are due to protein or ligand impurities,
Alternatively, it can be considered that these materials are due to water absorption.

Although high purity materials are desired, lower grade materials may also be used. The above materials have been found to be suitable.

(B) Effect of pH on binding reaction The fluorescence of riboflavin in the presence and absence of RBP (the ratio of riboflavin to RBP is 1: 1.3)
The pH was measured at 2 to 7. The results shown in FIG.
The change in fluorescence at pH below 0 is not dependent on RBP, whereas the change at pH above 3 clearly indicates that it depends on the degree of riboflavin binding to RBP. The pKa of the change at a pH above 3 was calculated to be about pH 3.7.

Accordingly, the pH at which the biomolecular switch containing RBP is operated is adjusted around pH 3.7.

Immobilization of RBP on silanized glass was performed using reagents such as, for example, triazine, carbodiimide or glutaraldehyde, resulting in a protein that retained reversible riboflavin binding activity. RB
P is derived from egg white and STC Laboratories (Winnipeg,
(Canada). Riboflavin was derived from yeast and supplied by Sigma Chemical Co. (Poole, Dorset, UK).

(C) pH adjustment of ligand binding using polypyrrole electrode Indium tin oxide coated with polypyrrole (IT
The pH was electrochemically controlled by the electrode O). The use of a polypyrrole coating on the working electrode avoids the evolution of oxygen and hydrogen which leads to a change in pH at both the working and counter electrodes. The use of a polypyrrole-coated electrode for pH adjustment is shown in FIG.

The indium tin oxide (ITO) electrode coated with polypyrrole is the working electrode, which is cut to a size of 8 mm × 50 mm to fit the inner wall of the measuring cell of the fluorometer. The counter electrode is a 9 mm x 9 mm mesh platinum which provides a large surface area. 0.1 M polypyrrole
It is electrodeposited from an aqueous solution of pyrrole and 0.1 M hydrochloric acid on an ITO electrode using a galvanostat (1 mA / cm ² ). Electrodeposition typically lasts for 5 minutes. To remove oxygen from the polypyrrole solution, oxygen-free nitrogen is passed through the solution prior to performing the electrodeposition. PH using these devices
To be able to adjust the pH, the ITO electrode (working electrode) coated with polypyrrole, the reticulated platinum electrode (counter electrode) and the Ag / AgCl reference electrode were immersed in a 1M aqueous KCl solution. To this solution was also passed through a nitrogen containing no oxygen to remove O ₂ and CO ₂ in advance. The change in pH was monitored using an ultrathin rod-shaped pH electrode. Next, the potential of the working electrode was set to +0.5 using a potentiostat.
V was varied between V and -0.7V. FIG. 5, which shows a typical cyclic voltammogram of a polypyrrole-coated electrode in aqueous solution, shows that relatively reversible anion uptake and release has occurred.

The pH can also be adjusted by physically (positionally) separating the working electrode and the counter electrode (that is, separating the working electrode chamber and the counter electrode chamber). In this case, the potential of the working electrode was maintained higher than the oxidation-reduction potential of riboflavin so that the oxidation-reduction reaction of riboflavin did not occur. Further, the liquid in the working electrode chamber is separated from the bulk solution, and the incident light (EL) impinges only on the liquid in the working electrode. FIG. 14 uses the arrangement of the electrode chambers as described above,
Fig. 3 illustrates a fluorescence spectroelectrochemical cell system used when BP and riboflavin are dissolved in a solution. The working electrode (platinum mesh) 1 was placed in a cuvette inserted upside down. Counter electrode (platinum mesh)
2 and a reference electrode (Ag / AgCl) 3 were placed in a Teflon container containing the bulk solution. The lid can be rotated to adjust the incident light (EL) angle to minimize scattering.

For immobilizing RBP and / or riboflavin, the electrochemical cell system shown in FIG. 15 was used. Working electrode (platinum mesh) 4 and immobilized R
BP aminopropyl glass beads 5 were placed in the working electrode chamber 6 at the center of the cell. The solution in the working electrode chamber is separated from the bulk solution electrically connected through the partition supported by the O-ring 7 and the sintered glass 8. The working electrode chamber has a depth of 0.8 mm. The depth can be changed to 2 mm by removing the spacer 9 just below. A groove 10 is provided at the bottom of the cell. A counter electrode (platinum mesh) 11 and a reference electrode (SCE) 12 are located in the bulk solution. The cover glass is fitted in the center of the cell window (top of the working electrode chamber). Incident light (excitation light: E
L) to the working electrode chamber 6 and the fluorescence (F
L) changes were observed.

According to the above method, the pH was adjusted by ± 0.2 units as shown in FIG. Such pH
Were used to control RBP-riboflavin binding. FIG. 8 shows the change in riboflavin fluorescence under the condition of the voltage-dependent pH cycle.

Example 2 Sulfate and Phosphate Ion Binding Proteins These proteins were obtained from E. coli and
Periplasmic space (per) of Gram-negative bacteria such as S. typhimurium
present in the iplasmic space) and form part of the nutrient intake system of these organisms. A variety of molecules, such as carbohydrates, amino acids and anions (eg, phosphate and sulfate) are taken up by these binding proteins. Many examples of these proteins are known,
They all have a common basic structure called "bean beans". When the ligand binds, the two round protrusions in the molecule approach each other and embed the ligand. This structural change can be observed by X-ray diffraction, but more importantly in this example, it can also be observed by fluorescence spectroscopy.

(A) Purification and Labeling Phosphate ion binding protein was obtained from E. coli, and sulfate ion binding protein was obtained from S. typhimurium, according to methods described in the literature (N. Medevsky, H. Rosenberg, Biochim.
Biophys. Acta 211 158, 1970; and ABPardee, JB
iol. Chem. 241 5886, 1966).

A series of fluorescent derivatives of this protein
Prepared using the following fluorophores; fluorescein isothiocyanate to label amino groups; acetamide derivatives for cysteine and methionine; and 7-chloro-4 to label tyrosine and lysine.
-Nitrobenzo-2-oxa-1,3-diazole. The intrinsic fluorescence of tryptophan residues can also be measured.

To determine the response of the various fluorescent derivatives to ligand binding, detailed spectrofluorimetric studies of these derivatives were performed.

(B) Immobilization FIG. 7 shows a configuration of a biomolecule switch according to the present embodiment.

First, a platinum metal film was formed on quartz by sputtering so as to function as an electrode for adjusting the anion concentration.

Next, specific fluorescent derivatives of each of the phosphate ion binding protein and the sulfate ion binding protein are immobilized on the quartz substrate 13. This quartz substrate 13
Has previously been silanized according to the method of Angew. Chem. (Int. Ed.) 25 236, (1986) by U. Deschler, P. Kleinschmit and P. Pastner.

Immobilized fluorescently labeled protein 14
Was then re-evaluated using a fluorescence microscope, including remeasurement of binding to the ligand.

(C) Redox polymer film The metal film acting as an electrode was coated with polypyrrole by electrodeposition using a galvanostat to form a polymer film electrode 15. The conductive polymer of polypyrrole exists in two charged states depending on the applied potential. In the reduced form, the polymer is electrically neutral and does not bind ligands (phosphate and sulfate). On the other hand, when oxidized, the polymer becomes positively dielectric and binds the ligand. Ligands for either phosphate and sulfate ions are present at known concentrations in the sequence of the protein. The increase in voltage applied on the protein sequence is used to form a gradient of ligand concentration on the protein sequence, thereby controlling the degree to which individual protein molecules come into contact with the ligand. Ligand is -0.6
At a potential of -0.9 V from -0.1 to +0.
It is taken in at 3 V (all voltages are relative to a saturated calomel electrode). The individual protein molecules act as switching elements and the excitation light (EL) produces a fluorescent output (FL) from the protein sequence.

The biomolecular switch according to the above embodiment can be used as a conversion algorithm or a memory.

FIG. 9 illustrates a generalized schematic biosensor according to one aspect of the present invention. In this biosensor, an immobilized protein sequence is used as an input sensor. The input here can be, for example, an unknown concentration of ligand, or a change in pH.

In the above, the present invention has been described based on two embodiments. However, various modifications can be easily made, and the principle applied to the above embodiment is applied to other biopolymers. Obviously gaining.

For example, adjustment of pH can be easily achieved using gold or platinum electrodes. Further, the above embodiments include:
Although attention is directed to homogeneous biomolecular switches in that a single polymer is used, it is clear that more than one polymer / label combination can be used. However,
For example, it may be necessary to identify multiple labels based on differences in fluorescence.

A specific example of a biosensor using a riboflavin biopolymer switch is described in detail in the Examples below.

Example 3 Riboflavin Biosensor The release of riboflavin from an immobilized RBP system is used as a biosensor to measure riboflavin since the binding protein produces an output signal that is responsive to a ligand concentration gradient. obtain. For this purpose,
Riboflavin binding protein (RBP) was purchased from Sigma Chem.
Immobilized on aminopropyl-regulated pore glass beads (pore size, 17 nM) from ical Co. using the following method: (1) Beads were dichloromethane, then acetone, ethanol, mili- Wash three times in Q water and diluted surfactant (one wash for 2 minutes) and then mili-Q
Rinse thoroughly with water.

(2) The beads were placed in 25% ethanol for 3 hours.
2 times, then 3 times in absolute ethanol (1
(2 minutes for 2 washes).

(3) The beads were then incubated in sodium-dried triazine solution in toluene at 50 ° C. for 4 hours (for 300 mg of beads, 4
50 μl of 2,4-dichloro-n-propoxy-s-triazine (manufactured by Lancaster Synthesis) 15 ml toluene).

(4) The beads are washed five times with toluene,
And it dried under nitrogen atmosphere.

(5) The beads were then washed with a pH of 0.1
Incubated with 4 mg / ml RBP in M phosphate buffer at 4 ° C overnight to 2 days with mixing on a rotary mixer.

(6) Beads were washed at least 5 times with PBS
(0.1 M phosphate buffer containing 1.37 M NaCl and 0.027 M KCl), then PBS-tw
een (containing 0.05% tween 80) and P
Washed three times each with BS.

(7) The beads were mixed with 1 M ethanolamine (pH 9) (or 0.1 M Tris / HCl, pH
8 could be used) and kept for 2 hours to overnight. (8) The beads were washed three times with PBS.

Different amounts of riboflavin were mixed with RBP-immobilized aminopropyl glass beads. Next, the bound riboflavin was released by the following procedure.

(A) Construction of Electrochemical Cell An electrochemical cell was constructed in a crystal dish 16 as shown in FIG. Immobilized RBP aminopropyl glass beads 17
With glass fiber sintered on the bottom (sintered glass)
18 was placed in a glass column. A working electrode (platinum mesh) 19 was embedded in the beads. A V-shaped notch 20 is provided at the bottom of the column extension to contact the bulk solution and remove any air trapped by tube insertion. The column containing the extension is supported by a socket 21. Counter electrode (platinum mesh) 2
2 and a reference electrode (SCE) 23 are located in the bulk solution.

(B) Ex situ (not original site) measurement I
(Ripoflavin binding to immobilized RBP controlled by potential) Riboflavin does not bind to immobilized RBP in an acidic solution. However, when a negative voltage (vs. SCE) is applied, pH
And riboflavin binds to the immobilized RBP. The riboflavin-immobilized RBP complex is then transferred to an acidic solution, releasing riboflavin and measuring its fluorescence ex situ with a fluorimeter.

First, the change in pH is measured in the following manner. Working electrode 19 was embedded in immobilized RBP aminopropyl glass beads 17. The level of the bulk solution (5 μM riboflavin solution, in 0.1 M KCl, pH 3) was
The column was set so that the volume became 0.3 ml.
pH measurements are taken when the potential is open circuit. When the potential was -0.2 V (vs. SCE), the pH change was very small. It took 30 minutes for the pH to increase from 3.0 to 3.5. When the potential is held at -1.0 V,
Near the aminopropyl glass beads, the pH value changed from 3.0 to 6 or more in 5 minutes. And, after the solution in the column was stirred, it was 4.0. At this potential, riboflavin can be reduced, followed by a change in riboflavin fluorescence. The mobility of hydrogen ions is
Because the mobility of riboflavin is much faster, the effect of pH on the change in fluorescence can dominate the reduction of riboflavin up to 5 minutes. To prove this assumption, 5-10
After applying a potential at -1.0 V for minutes, the acidic solution (p
In H3), riboflavin was released and the fluorescence of the released riboflavin was measured. If the redox reaction dominates the pH change, no fluorescence is observed.
However, fluorescence due to the released riboflavin was observed, and thus the results showed that the effect of the redox reaction was not critical in this assay format.

A 5 μM riboflavin solution (pH 3 0.1
(In MKCl) was sparged with nitrogen for at least 10 minutes. The solution was poured into a crystallization dish 16 and the solution level was adjusted to fill the glass column to 0.5 ml. Here, the working electrode 19 and the immobilized RBP aminopropyl glass beads 17 are arranged at predetermined positions in advance. The potential was kept at -1.0 V for 5 minutes. The working electrode was then immediately removed, and the glass column was connected to a vacuum pump to remove excess solution in the column. The dried glass beads were then immediately squeezed to a pH of 0.3.
Resuspended in 1 ml of 1M KCl solution and transferred the mixture to a cuvette. Two minutes later, after the beads had settled, the fluorescence of the released riboflavin was measured. Immobilized BSA (bovine serum albumin) on aminopropyl glass beads was used as a control, as removal of the solution by a vacuum pump was not sufficient to remove all unbound riboflavin. The presence of BSA was 10% relative to riboflavin.
It has been previously confirmed that a 0-fold molar excess does not affect riboflavin fluorescence. Next, riboflavin fluorescence was measured for immobilized BSA in the same manner as used for immobilized RBP (see table below).

[0067]

[Table 1]

[0068] Riboflavin was released from the immobilized protein on aminopropyl glass beads. At pH 3,
After a potential of -1 V was applied in a 5 [mu] M riboflavin solution, the immobilized protein was dried and adjusted to pH 3 at 0.
Resuspended in 1M KCl solution. After the aminopropyl glass beads settled, the fluorescence of riboflavin in the supernatant was measured (excitation at 455 nm, emission at 525 nm). For each protein, three separate samples were measured. The difference between RBP and BSA (control) is significant. Thus, the results indicate that the binding of free riboflavin to immobilized RBP is controlled by external stimuli, ie, the applied potential.

(C) Ex situ measurement II (Riboflavin release from immobilized RBP controlled by potential) Riboflavin bound to immobilized RBP has a positive potential (vs. S
It can be released by a pH drop resulting from the application of CE). The released riboflavin can then be measured ex situ.

The immobilized RBP aminopropyl glass beads were first mixed with a 5 μM riboflavin solution at pH 5 to form a riboflavin-RBP complex, and then mixed with a pH 5. 0 to remove excess riboflavin.
Washed with 1M KCl. The beads were placed on a glass column and the electrodes were set as described above. A 0.1 M KCl solution at pH 5 was poured into the crystallization dish until the column was filled with 1 ml of solution. At 2 V, it took 10 minutes or more for the pH value to be 4 or less. Next, the working electrode was removed.
The column was also removed from the crystallization dish and allowed to stand for 1 minute until the beads settled. Subsequently, the fluorescence of the supernatant was measured with a fluorimeter. Fluorescence from the released riboflavin was clearly observed.

When a control immobilized BSA was used, BS
Since A does not bind to riboflavin, all riboflavin was removed during the washing step before applying the potential. Thus, no fluorescence was observed in the supernatant of BSA immobilized on aminopropyl glass beads. This result indicates that riboflavin is released from the immobilized RBP by an external stimulus, ie, application of a positive potential. Since this procedure can be applied to a riboflavin biosensor, a quantitative measurement of riboflavin release controlled by this potential is described below.

(D) Biosensor In the riboflavin biosensor, the applied potential was changed to 4 V (vs. SCE) in order to increase the measurement speed. 4V, 5 minutes was sufficient to release riboflavin. FIG. 11 shows that the fluorescence intensity of riboflavin released by application of a potential is proportional to the riboflavin concentration in the sample. The fluorescence intensity of the released riboflavin is 45
Excited at 5 nm and then measured at 525 nm.

For a sample lipoflavin concentration of 1-7 μM, a linear relationship was observed. Increasing the total amount of immobilized RBP increases the upper measurement limit. The lower limit is improved by increasing the electrode surface area adjacent to the immobilized RBP beads to make the pH change more effective.

As shown in FIG. 12, immobilized RBP2
The glass column 25 holding the aminopropyl glass beads having No. 4 and the working electrode (Pt electrode) 26 are connected to a tube so that the sample solution flows in the direction of arrow A, and the column functions as a flow cell and operates as a detector. To enable continuous measurement of sample riboflavin concentration.

As the sample solution passes through column 25, riboflavin selectively binds to the immobilized RBP column.
Next, hydrogen ions are generated by applying a positive potential to the working electrode to lower the pH and release riboflavin. The fluorescence is then measured with a fluorescence detector. The column (sensor) is already ready to measure the next sample.

RBP can also be immobilized on platinum and ITO electrodes, thereby eliminating the need for the aminopropyl glass bead column described above. RBP
Is a triazine chemistry (Mehtods in Enzymology (vol. 4)
4, 1976, Weetall HH)).

The biosensors developed using this system are reagent-free and, more importantly, reproducible in situ and are therefore reversible. Selectivity can be further improved by using the action of the riboflavin redox reaction on fluorescence if the potential is scanned by another electrode on the detection side. In this way, the presence of interfering substances that bind non-selectively to the column can be identified.

Since riboflavin is a vitamin B2, this biosensor is useful for measuring its concentration in urine and blood samples.

Although the present invention has been described by way of examples, various modifications can be readily made, and the principles used in the examples can be applied to other biopolymers. In addition, the above examples relate to using a single polymer,
Although focusing on homogeneous biopolymer switches, for example, based on labels with different fluorescent properties,
While it is necessary to distinguish labels, it is clear that more than one polymer / label combination can be used.

Although the electrodes described in the above examples consider only one-dimensional spacing, the fact that macromolecules can detect a two-dimensional concentration profile, for example, as shown in FIG. It is clear that the advantage can be used to arrange the pH generating electrode 27 and the immobilized protein 28 two-dimensionally.

The two-dimensional concentration gradient (profile) controlled by the applied potential can be observed using a fluorescence microscope. FIG. 16 shows an example of detection of a two-dimensional ligand concentration profile. FIG. 16 shows the results of observing riboflavin released from RBP by applying a potential by using a fluorescence microscope. The applied voltage is +2 V (vs. Ag / AgC) for 10 seconds.
In l), it was then switched to -1V. Riboflavin fluorescence was measured using a filter (excitation between 450 and 490 nm, 515
発 光 585 nm emission) and images were taken every 2 seconds with a CCD video camera. In the figure, a is a gray scale image, and b is a pseudo color image.

The pH generating electrodes 27 can be arranged two-dimensionally,
The polymer can then be immobilized over the rest of the region by a molecular valediction technique such as photolithography. The arrangement of pH electrodes offers the possibility of complex input patterns. Each of the immobilized macromolecules 28 interacts with each other in the local and remote areas by a concentration gradient pattern, thereby producing a fluorescent output pattern.

In this arrangement, a group of immobilized macromolecules is affected not only by directly adjacent electrodes but also by electrodes at distant sites. Thus, on a short time scale, the macromolecules are strongly affected by the electrode closest to them, while on a long time scale, effects due to interaction with distant electrodes also occur. In this way, this electrode arrangement allows for complex input patterns and enhanced performance with many molecules affected in a parallel fashion depending on the distance from the electrode site and the time scale of the stimulus input. To provide sex.

In this way, the immobilized polymers interact with each other in the region where they are located. The binding state of each molecule affects the binding state of nearby molecules, and is itself affected. Distant molecular sites are also interacted by the effect of the concentration gradient pattern. Due to the complexity of the input possibilities promoted by the interacting two-dimensional array structures described above, there is a degree of flexibility in the form in which the inputs can appear, and complex and simpler patterns are possible. .

In the above example, an electrochemical method was employed to generate the response in the system. However, it is also possible to use optical techniques for modulation of the pH.
For example, rapid pH modulation can be achieved with laser pulses.

In classic von Neumann theory, a question in the form of an input is received, processed step-by-step by a series of independent logical operations, and arrive at an answer. This type of method is critically dependent on the type of conventional logical connection and operation, and the system considered by biomolecular switches is not possible.

Computers using the biomolecular switch of the present invention operate in different ways. The output from this new switch is expressed as a two-dimensional binding interaction mediated by a pH gradient, and is in fact a direct result of the input itself. That is, the presented question generates a condition under which an answer is given as a two-dimensional matrix.

The programming of this system can be facilitated by a change in the dynamics of the binding affinity, followed by the use of genetically engineered macromolecules, or by the use of different macromolecules, each having a different affinity for this macromolecule. Achieved by the use of ligand analogs.

As described above, the present invention provides a biomolecular switch utilizing the ability of a biopolymer to convert between a plurality of states in response to a physical or chemical change in its microscopic environment. I will provide a. Such a biomolecule switch can contribute to improvement of integration and miniaturization of information processing devices and the like.

[0090]

[Brief description of the drawings]

FIG. 1 shows riboflavin binding protein (RBP)
1 is a schematic diagram showing the binding of riboflavin to riboflavin.

FIG. 2 is a graph showing RBP binding activity.

FIG. 3 is a graph showing the effect of pH on the binding reaction.

FIG. 4 is a conceptual diagram showing adjustment of pH using a polypyrrole-coated electrode.

FIG. 5 is a graph showing a typical annular voltammogram of a polypyrrole-coated electrode.

FIG. 6 is a graph showing pH adjustment using a polypyrrole-coated electrode.

FIG. 7 is a schematic diagram showing a biomolecular switch of the present invention.

FIG. 8 is a graph showing changes in riboflavin fluorescence under conditions of voltage-dependent cycling.

FIG. 9 shows a generalized schematic of a biosensor according to the present invention.

FIG. 10 shows an electrochemical cell suitable for measuring riboflavin binding and release.

FIG. 11 is a graph showing the fluorescence intensity of riboflavin released from immobilized RBP by applying a positive potential.

FIG. 12 is a diagram showing a flow cell of immobilized RBP having a platinum electrode.

FIG. 13 is a diagram showing an RBP biopolymer switch in which pH generating electrodes are two-dimensionally arranged.

FIG. 14 is a diagram of a fluorescence spectroelectrochemical cell system used when RBP and riboflavin are dissolved in a solution.

FIG. 15 is a diagram of an electrochemical cell system used for immobilizing RBP and / or riboflavin.

FIG. 16 is a diagram showing an example in which RB is applied by applying potential;
In the fluorescence microscope image obtained by observing riboflavin released from P, a shows a gray scale image and b shows a pseudo color image.

[Explanation of symbols]

 Reference Signs List 1 working electrode 2 counter electrode 3 reference electrode 4 working electrode 5 immobilized RBP aminopropyl glass beads 6 working electrode chamber 70 ring 8 sintered glass 9 spacer 10 groove 11 counter electrode 12 reference electrode 13 quartz substrate 14 immobilized protein array 15 Polymer membrane electrode 16 Crystal dish 17 Immobilized RBP aminopropyl glass beads 18 Sintered glass 19 Working electrode 20 V-shaped notch 21 Socket 22 Counter electrode 23 Reference electrode 24 Immobilized RBP 25 Glass column 26 Working electrode (Pt electrode) 27 pH generation Electrode 28 Immobilized polymer

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-3-171686 (JP, A) JP-A-63-237584 (JP, A) JP-A-2-281770 (JP, A) JP-A-6-237 291303 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) H01L 51/00 G01N 21/76 G01N 27/30 C12N 11/00-13/00

Claims (6)

(57) [Claims] 1. A biomolecule switch suitable for use in a biosensor or data acquisition and processing device, comprising: an array of biopolymers immobilized on a carrier, each polymer comprising: Can exist in two or more states that can be reversibly interconverted, and between each macromolecule in the sequence,
An array of biopolymers that can interact; an input means that can selectively apply a stimulus to the polymer to convert at least some of the polymer from a non-stimulated state to a stimulus-dependent state And an output means for measuring or monitoring a change in the state of the macromolecule. 2. The biomolecule switch according to claim 1, wherein said biopolymer is a protein, nucleic acid or polysaccharide. Wherein said input means, electrical or electromagnetic means pH or temperature or ionic strength Doma other in microscopic environment of the polymer acts to modulate ligand concentration or any combination thereof The biomolecule switch according to claim 1 or 2, wherein 4. The biomolecule switch according to claim 3, wherein said output means is adapted to measure a relative quantity of said non-stimulated state and said stimulus-dependent state. 5. The method of claim 2, wherein the input acts to form a ligand concentration or pH gradient on the biopolymer sequence;
4. The biomolecule switch according to claim 3, wherein said output means is adapted to monitor a reaction pattern of said polymer reflecting a change in a pattern of a given stimulus. Wherein said output means nuclear magnetic resonance, infrared spectrum, UV difference spectra, using fluorescence, chemiluminescence or bioluminescence biomolecules switch according to any one of claims 1 to 5.