TWI473997B - Electrosensing antibody-probe detection and measurement sensor having conductivity promotion molecules - Google Patents

Description

Electro-sensing antibody probe detection and measurement sensor with conductive trapping molecules

The present invention is generally directed to detection and measurement using electrosensing using an antibody as a probe. In particular, the present invention relates to sensors and related methods that can be used for electro-detection antibody probe detection and measurement.

In medical and related fields of application, it is known to use biochips to detect target substrates in a test sample. Biochip sensors (or biosensors) have been applied to the detection of specific target substances under considerations such as accuracy and cost. If the technology is feasible and cost-effective, biochip sensors are clearly more useful for any imaginable use, in addition to detecting the presence or absence of target substances, if further quantitative measurements are made. . For example, in biomedical applications, if, for example, from 1 to 10, 1 to 100, or even higher resolution, and maintaining accuracy, the measurement indicates that the target substance is present in the sample. The degree scale is obviously very informative for the purpose of its original sensing application.

Biochips based on optical sensing technology are common to today's global biosensing technology. Such wafers rely on the need for optical sensing to capture the results of the detection reaction on the wafer using a large, bulky and expensive precision optical instrument. In order to avoid these problems, the biochip using the principle of electro-pressure is obviously a reasonable method of miniaturization and low cost. Among the techniques of electro-sensing wafers, after the wafer is placed in the test sample, its inspection (or sensing) is an electrical inspection. Sensed by the sample to be detected may be impedance, capacitance, resistance, conductance, current, or any other useful electrical parameter. .

However, until now, the technology of electrical biosensing has its limitations, which is due to the fact that most liquid bioassay samples are not electrically conductive. 3A and 3B illustrate how conventional electro-sensing techniques are not suitable for antigen detection using antibody probes. For example, FIG. 3A schematically illustrates a conventional technique for sensing a wafer 300 using, for example, an antibody molecule immunoglobulin G (322) immobilized on the surface of both the positive electrode 312 and the negative electrode 314 of the wafer, wherein the electrode can be For example, a metal film of gold (Au), silver (Ag), copper (Cu) or nickel (Ni). To have a practical use, the system must be capable of permitting the measurement of the environment generally indicated by reference numeral 305 in the figure, i.e., sensing the amount of change in current between the two electrodes of wafer 300.

However, after the introduction of the sample to be tested, after the antigen molecule 332 therein (which is mostly a non-conductor or a poor conductor in nature) interacts with the antibody molecule 322 immobilized on the surface of the electrode, The electrical conductivity between the electrodes of this sensing system is still extremely poor, as shown in the schematic of Figure 3B. Therefore, the electro-sensing wafer technology of the prior art is only applicable to a biochip which uses an enzyme or a catalyst as a probe to perform a redox reaction to generate a current-voltage change, and its application is extremely extreme. Limited.

Accordingly, it is an object of the present invention to provide a sensor for detecting and measuring an electrosensing antibody wafer for detecting the presence of various target substances.

The object of the present invention is also to provide an electrosensing antibody probe for detecting and measuring different degrees of expression for detecting the presence of various target substances and the amount of various target substances.

Another object of the present invention is to provide a simple, small, and low-cost electro-sensing antibody probe detection and measurement sensor to replace large, high-precision, and expensive hardware devices.

Another object of the present invention is to provide an electrosensing antibody probe detection and measurement sensor which can be used for detecting various target substances and can be widely applied to biomedical products. Domains such as environmental control and industry.

To achieve the above and other objects, the present invention provides a mercapto-oligothiophene conductive eliciting compound which is capable of detecting and measuring a probe capable of supplying a test antibody. The electrosensing sensor comprises a second electrode and a layer of antibody immobilized on the surface of at least one of the electrodes. A conductively-exposed molecule is attached to and/or distributed between the electrodes to which the antibody is immobilized to enhance electrical conductivity between the electrodes. In a particular aspect, the antibody probe molecule of the electrosensitized wafer of the present invention and related methods can be said to be "wearing a tight layer with electrical conductivity", which makes electrical conductivity in the system. Essentially becomes "amplified" to the extent that the instrument used today is sufficient for accurate sensing. The measurable electrical parameters of the electrical sensing wafer, such as resistance values, thus become not only detectable but also readable, which thus becomes a meaningful parameter for interpretation.

The present invention provides a sensor for powering a sensory test sample, comprising two electrodes electrically disconnected and substantially separated from each other; and a layer of antibody immobilized on a surface of at least one of the electrodes, The antibody has specific binding reactivity with respect to the antigen; and a conductive attracting molecule covalently bonded to the antibodies to enhance the conductive properties between the electrodes. When the antigen present in the test sample mixed with a buffer is contacted with the antibody-immobilized electrode, the antibody captures the antigen and thereby changes the electrical conduction characteristics between the electrodes, wherein The amount of change provides a qualitative and quantitative indicator of electrosensitivity of the antigen.

The present invention utilizes the electrical conductivity of the sensing wafer system (the wafer environment and the antibody molecules itself that react with it) to achieve a practical use of the inductance measurement. In particular, the antibody probe molecule of the sensing wafer of the present invention and related methods can be said to be "wearing a tight-fitting electric conductive body", which makes the electrical conductivity in the system substantially become The instrument that is "zoomed in" to the application of today is sufficient for accurate sensing. Sensitive measurement of a wafer such as a resistance value Sexual parameters, so that not only become detectable and can be interpreted, it becomes a meaningful parameter for interpretation.

According to the present invention, an antibody immobilized on a sensing wafer as a detecting probe is substantially converted into a semiconducting or even a good electrical conductivity by its non-conducting nature. This allows the value of the electrical impedance change of the target substance in the sample liquid to be detected (after it contacts and reacts with the antibody on the sensing wafer), which can be detected not only by the instrument but also with sufficient accuracy. Test discrimination. The measured readings can therefore be applied to the interpretation of the intended target substance detection.

In fact, as can be understood by those skilled in the art, in addition to the impedance value, other electrical parameters such as the capacitance value of the system are also improved by the present invention by improving the electrical conductivity of the entire system. Measure. Moreover, in addition to interpreting the rigorous definition of the reciprocal of the resistance value, the term "conductivity" is more broadly interpreted within the scope of the invention as the electrically conductive state in its system. Therefore, the term "conductivity induced" should be interpreted here as "the enhancement of the electrical conduction state".

Therefore, the sensor and method of the present invention can establish an environment of electrical conduction, which can allow the change of electrical conductivity caused by the appearance of the captured target substance in the environment, and can be detected not only Measured and can be measured and interpreted. Since the sensor and method of the present invention substantially amplifies the electrical characteristic detection range of the entire test sample system, any variation in electrical properties, whether impedance, current or capacitance, whether it is DC or DC ) or any selected frequency of alternating current (AC) measurements are easily detectable and accurately quantified. The amount of change can thus be a quantitative indicator of the amount of target substance present in the sample being tested.

Figure 1 shows the architecture of a basic electro-sensing system of the present invention. The sensing wafer 100 constructed on a substrate 110 has an entire layer of antibody probes 120 fixed on the surfaces of its positive electrode 112 and negative electrode 114, wherein the electrodes may be, for example, Au, Ag, Cu or Ni, etc. Metal film. Electrodes 112 and 114 serve as substantial substrates for antibody probes selected for immobilization to a particular antigen.

An embodiment of the novel electro-sensing technique of the present invention based on the electro-sensing wafer 100 can be combined with a detection circuit and a fluid system to provide a sensing chamber 102. Within this cavity, the test sample is brought into contact with the wafer to allow the target antigen molecule 134 suspended in the liquid sample to become the antigen 132 captured by the antibody probe 120.

As will be explained in more detail below, the system of Figure 1 allows accurate measurement of the concentration of target antigen in the test sample. This is measured by applying a voltage between the electrodes of the sensing wafer and passing through a current measuring instrument, as shown in the figure.

2A and 2B show two possible configurations of an electro-sensing wafer in accordance with a preferred embodiment of the present invention. The sensing wafer 200A of FIG. 2A is in the form of a typical flat wafer, with sensing electrodes 212A and 214A disposed side by side on its substrate 210A. Such a flat sheet-shaped wafer structure relies on the cooperation of its corresponding wafer reading processing device to form a sample cavity in which sensing can be performed.

In contrast, the electro-sensing wafer 200B of FIG. 2B has a tubular configuration in which the two sensing electrodes 212B and 214B are disposed facing each other on the inner surface of the tubular "substrate" 210B. on. With such a tubular configuration, as long as the wafer is inserted into its corresponding processing reading machine to close its ends, the sensing wafer 200B can easily obtain a sample cavity 202B.

4A-4C respectively illustrate the preparation of a preferred embodiment of a sensing wafer in accordance with the present invention and the sensing of a sample thereof. It is noted that the electrodes, antibodies, antigens, and conductive attracting molecules shown in the figure are not shown in the correct scale. In order to facilitate the explanation of the present invention, some of the figures shown in the drawings are shown in an exaggerated proportion.

Figure 4A shows the basic system of a sensing wafer of the present invention in which the overall conductive properties of an electrically conductive environment are enhanced by the use of conductive attracting molecules. In a preferred embodiment, the film-formed Au is used to form substantially positive and negative electrodes 412 and 414 on the substrate 410 of the sensing wafer 400. Metals such as Ag, Cu, and Ni are also utilized in the present invention to fabricate electrodes. Suitable alloys, for example, indium tin, depending on the application Oxide, ITO), can also be used.

The electrically conductive molecule 442 is bonded to the electrode, as shown in the figure, which is attached to the electrode surface. According to the invention, such molecules thus form conductive attracting molecules immobilized on the surface of the electrode. When the wafer of the present invention is used, this allows the basic sensing system of the present invention to provide an enhanced electrical conduction environment, since the conductive attracting molecules modify the surface properties of the sensing wafer, As a result, the electrical conductivity of the bare sensing wafer system is improved. That is, after the antibody probe molecules are present in the wafer system, the conductivity between the positive electrode and the negative electrode is greatly enhanced. Because of the greatly improved electrical conduction environment between electrodes 412 and 414 as indicated by 405A in the figure, the system can sense the change in current before and after the presence of the antigen between the electrodes of sense wafer 400.

Substances suitable for use as conductive attractants include, but are not limited to, oligothiophene-silane, oligothiophene-thiol, 1-phenyl oligothiophene ((1-phenyl)- Oligothiophene), 2-phenyl-oligothiophene, side-arm oligothiophene, oligophenyl oligothiophene, and derivatives thereof.

In FIG. 4B, antibody 422 for probe use of a particular target antigen is applied to the layer of conductively induced molecules of the sensing wafer 400 and is covalently bonded thereto. Because of this layer of immobilized antibody, at this stage (ie, when the target antigen has not yet appeared), the conductivity of the sensing wafer is somewhat reduced in this already electrically conductive environment 405B, but is still easy to use. The instrument is within the range of measurement.

When antibody 422 is present, wafer 400 in Figure 4B forms an electric antibody sensor that is ready for electro-sensing applications for its particular target antigen molecule. For any predetermined sensing application, the corresponding specific non-conductive antibody molecule needs to be first immobilized on the wafer. For example, an immunoglobulin G molecule can be used as an antibody probe for detecting, for example, S100, alpha-fetoprotein, and tropolin I. The overall electrical conductivity of the system will be reduced, The extent reflects the fact that the probe is present in the system. This change in electrical conductivity becomes the reference reference value at the time of detection.

Fig. 4C shows a case where electrosensing is performed by exposing a target antigen to a probe antibody immobilized on a wafer. The wafer 400 in Figure 4B in which the probe has been prepared for sensing is exposed under a test sample. Since the probe antibody 422 that can be detected for a specific target has been immobilized on the wafer, the target present in the sample, that is, the antigen 432, is captured by the antibody or, in other words, interacts with the antibody. (interaction) reaction.

As the captured antigenic molecules 432 appear in the system, the overall conductivity of the entire electrically conductive environment 405C changes further (i.e., compared to Figure 4B), and its electrical impedance measurement reads. The change (i.e., measured as the current between the electrodes) becomes an indicator of the extent of the amount of antigen present in the system.

According to the electro-sensing test performed by the present invention, when a sample containing a non-conductive target antigen is introduced into the fluid detection measurement environment provided by the sensing wafer of FIG. 4C, the overall electrical conductivity of the system is followed. The change is reduced. This reduction is reflected by the corresponding decrease in the measured current value. The degree of variation is proportional to the number of antigens that are captured by the wafer. However, it should be noted that in some cases, after binding of certain antigens in the test sample to the antibody probe, it may lead to conductance compared to the fact that the antigens have not appeared in the system before they appear in the system. The increase in degrees.

Figures 5A-5C respectively illustrate another preferred embodiment of sensing a wafer in accordance with the present invention, which is prepared and sensed for a sample. The examples depicted in Figures 5A-5C and those shown in Figures 4A-4C, except that they sense the physical configuration of the wafer, are positioned substantially identically with the electrodes facing each other. In accordance with the inference of the present invention (but the invention should not be limited to this inference), the arrangement of such electrodes may be due to their preferred conductive properties relative to the planar configuration of Figures 4A-4C. It is allowed to produce better sensing performance.

Figure 6 is a schematic diagram showing the practical use of the electro-sensing wafer and method of the present invention. The curve in the figure shows the conductivity of a test sample compared to the antigen concentration present in the sample. The relative relationship.

The vertical axis in Figure 6, i.e., the axis of electrical conduction properties, on the symbols A, B, C, D, D' and D", respectively, is the electrical conductivity of the sensing wafer at various stages during its preparation:

A: substrate

B: electrode

C: Conductivity induced

D, D', D": antibody probe addition

In order to perform a wide range of concentration measurement on the target antigen present in the sample, the conventional technique is in the small current measurement reading range (BD' or BD" in the figure, depending on the probe to be added. In the case of reducing or increasing the overall electrical conductivity, it is attempted to measure the conductivity reading of the sample. The reading range of the current value is too small to be practical, and it is difficult to distinguish whether the target substance exists or not. Acceptable read resolution yields an antigen concentration curve within the sample, E' or E".

In contrast, if a conductive attracting molecule is used according to the present invention, the detection range (BD) of the target substance is substantially enlarged in the sense of a certain level, which can thus allow a good resolution. In other words, the accuracy is better, and the target concentration is read. This is because, as clearly shown by the characteristic curve E in Fig. 6, regardless of whether the concentration of the target substance in the liquid sample environment is linear or nonlinear between the corresponding measured currents, the broad measurement range is corresponding. The target detection carried out inside can of course make the interpretation of the instrument readings easier.

Figure 7 illustrates a sensing wafer having antibodies immobilized by covalent bonding of conductively-exposed molecules, which enhance electrical conductivity throughout the detection system, in accordance with an embodiment of the present invention. In a preferred embodiment, taking the sensing wafer 700 of FIG. 7 as an example, the antibody molecules 722 are first adjusted for covalent bonding using conductive deriving molecules 742 prior to immobilization on the surfaces of electrodes 712 and 714. However, as will be appreciated by those skilled in the art, in another embodiment, antibody molecules 722 can be immobilized on the surfaces of electrodes 712 and 714, and then modified using conductive attracting molecules 742.

Unlike the case where the conductive attracting molecules 442 in the system of FIG. 4 are used as the bonding agent for the immobilized antibody 422 and the sensing wafer electrode 412/414, there are more than one conductive attractant in FIG. Ascending molecule 742 binds to each antibody molecule 722. This arrangement is applicable to antibodies which can be directly attached to the electrodes by being attached to the electrodes.

In contrast, in another preferred embodiment, for the system of sensing wafer 800 of FIG. 8, conductive attracting molecules 842 are also used to bond antibody 822 to electrodes 812/814. As noted above, these conductively induced molecules play a dual role in the system. Conductively induced molecules not only promote the electrical conductivity of the system, but also bind the antibody molecules and electrodes. In contrast to the system of Figure 4, the conductivity-exposed molecules 842 can be used to further enhance the electrical conductivity of the system when applied to the antibody.

Figure 9 illustrates the attachment of antibody 922 and conductive derivatory molecule 942 in more detail. An antibody 922 having a Y-type molecular structure as shown in the figure is structurally covalently bonded with a plurality of conductive deriving molecules 942. Such conductively induced molecules, such as the oligophenyl-thiophene and its derivatives depicted in the figures, are shown in the thiophene molecule 9421, modified by phenyl 9422, and covalently bonded thereto. Antibody 922. A portion of the conductive attracting molecule, 942A on the extended end of the Y-type molecular structure of antibody 922, can link antibody 922 to the electrode wafer while also improving overall electrical conductivity.

The present invention has been described in detail with reference to the specific embodiments thereof, and the various modifications, variations, and equivalents thereof are still possible. Therefore, the above description and related drawings are not to be construed as limiting the scope of the scope of the appended claims.

400,700,800‧‧‧Sensor wafer

405A, 405B, 405C, 505A, 505B, 505C 705, 805‧‧‧Electrically conductive environment

410,510,710,810‧‧‧ wafer substrate

412,414,512,514,712,714,812,814‧‧‧ wafer electrodes

422,522,722,822,922‧‧‧antibodies

432, 532‧‧ ‧ antigen

442,542,742,842,942,942A‧‧‧ Conductive inducers

Figure 1 shows the architecture of a basic electro-sensing system.

2A and 2B show two possible configurations of an electro-sensing wafer.

Figures 3A and 3B illustrate how conventional electrophysiological measurements are not suitable for antigen detection using antibody probes.

4A-4C respectively illustrate the preparation of a preferred embodiment of a sensing wafer in accordance with the present invention and the sensing of a sample thereof.

Figures 5A-5C respectively illustrate the preparation of a preferred embodiment of a sensed wafer in accordance with the present invention and the sensing of a sample thereof.

Figure 6 illustrates why the electro-sensing wafer and method of the present invention have substantial utility.

Figure 7 shows a sensing wafer containing an antibody immobilized by covalent bonding of conductively-exposed molecules, which enhances electrical conductivity throughout the detection system, in accordance with an embodiment of the present invention.

8 shows a sensing wafer according to an embodiment of the present invention, which comprises an antibody immobilized by covalent bonding of a conductive deriving molecule, and the surface of the antibody is also modified by a conductive derivation molecule, which can enhance the entire detection system. Electrical conductivity.

Figure 9 shows the attachment of an antibody to a conductive derivatory molecule.

800‧‧‧Sensor wafer

805‧‧‧Electrically conductive environment

810‧‧‧ wafer substrate

812,814‧‧‧ wafer electrode

822‧‧‧ antibody

842‧‧‧ Conductive inducers

Claims (6)

A sensor capable of supplying a test for detecting an antigen in a sample, comprising: a second electrode electrically disconnected and substantially separated from each other; a layer of antibody immobilized on a surface of at least one of the electrodes, the antibody being relative to The antigen has a specific binding reactivity; and a plurality of electrically induced molecules, wherein one end of each of the electrically induced molecules is a phenyl group and a derivative thereof, and are covalently bonded to the end via the end These antibodies are used to enhance the conductive properties between the two electrodes. The sensor of claim 1, wherein when the antigen present in the test sample mixed with a buffer is in contact with the antibody-immobilized electrode, the antibody captures the antigen and thus changes the two electrodes The electrical conduction characteristic between, wherein the amount representing the change provides a qualitative and quantitative indicator of electrosensitivity of the antigen. The sensor of claim 1, wherein the conductive attracting molecules are bonded to the antibodies by covalent bonding. The sensor of claim 1, wherein the layer of anti-system is coupled to the electrodes via the conductive attracting molecules. The sensor of claim 1, wherein the electrical sensing system is a current measurement between the two electrodes by direct current (DC) or alternating current (AC). The sensor of claim 1, wherein the electrical sensing system is a capacitance measurement between the two electrodes.