TWI522614B - Peacock Green Detector - Google Patents

Description

Malachite Green Detector

The present invention relates to a detector, and more particularly to a malachite green detector.

Malachite green is a triphenylmethane dye that has been widely used in aquaculture since 1930 to protect and treat infections in fungi and ectoparasites. However, malachite green will remain in the adipose tissue of fish. It is highly cytotoxic to mammalian cells and is also a promoter of liver tumors. It may cause harm to the immune system and reproductive system and may cause cancer. Therefore, malachite green has become a pole today. Controversial compounds, and many related detection methods have also been applied to detect malachite green.

The current commercial measurement methods include High Pressure Liquid Chromatography (HPLC) and high-precision experimental instruments such as liquid chromatography tandem mass spectrometry (LC/MS/MS). Although the malachite green content has its advantages, including low detection limit and high detection accuracy, its disadvantage is that the analytical instrument is expensive and bulky, and cannot be directly tested by the fish pond, and the trained personnel can operate the instrument. As well as long analysis time, although there is also an ELASA test kit for detecting malachite green, it cannot be implemented at point detection and still needs to be trained. Talent is operational, and although it takes less time to use precision instruments, it still takes about an hour. In recent years, researchers have attempted to directly detect malachite green using biochips made of acetylcholine. Although it has been convenient to directly test on farms, its detection sensitivity is insufficient and it still needs improvement.

Accordingly, it is an object of the present invention to provide a malachite green detector which is small in size and which is convenient for malachite green detection and has excellent detection sensitivity of malachite green.

Therefore, the malachite green detector of the present invention can be used to detect malachite green, and includes a sensing electrode, the sensing electrode includes: an insulating substrate, a sensing film coated on the surface of the substrate, and a fixed a conductive layer electrically connected to the sensing film, the sensing film includes a conductive layer coated on the surface of the substrate and electrically connected to the conductive line, and a cover is fixed on the surface of the conductive layer and is The malachite green reaction produces a signal of the butyl cholinesterase reaction layer.

The effect of the invention is that the sensing electrode design for fixing the butylated cholinesterase reaction layer through the conductive layer can not only be used to reduce the volume of the entire malachite green detector, but also is convenient to carry and use. The peacock green detection sensitivity, with a minimum detection limit of 10 ppb, has a very high commercial value.

2‧‧‧ Malachite Green Detector

3‧‧‧Sensing electrode

31‧‧‧Substrate

32‧‧‧Sensing film

321‧‧‧ Conductive layer

322‧‧‧ 醯 cholinesterase reaction layer

33‧‧‧Flexible wire

34‧‧‧Package

340‧‧‧ window hole

4‧‧‧ reference electrode

5‧‧‧Signal amplification module

51‧‧‧ non-inverting input

52‧‧‧inverting input

53‧‧‧ Signal output

800‧‧ ‧ specimen

900‧‧‧Data analysis equipment

901‧‧‧Signal Processor

902‧‧‧Analytical device

Other features and effects of the present invention will be described with reference to the drawings. It is clearly shown in the embodiment, wherein: FIG. 1 is a schematic structural diagram illustrating a preferred embodiment of the malachite green detector of the present invention coupled with a data analysis device; FIG. 2 is a schematic diagram of a manufacturing process illustrating the preferred embodiment. FIG. 3 is a partial cross-sectional view showing the structure of the sensing electrode of the preferred embodiment; FIG. 4 is a control group for detecting malachite green by acetylcholinesterase. The inhibition ratio of acetylcholinesterase activity is shown in Fig. 5; and Fig. 5 is a graph showing the inhibition ratio of cholinesterase activity in the experimental group of peacock green detection by butyl cholinesterase.

The invention will be further illustrated by the following examples, and it should be noted that in the following description, like elements are denoted by the same reference numerals. It should be understood that the examples are for illustrative purposes only and are not to be construed as limiting.

Referring to Figures 1, 2 and 3, the Malachite Green Detector 2 of the present invention is suitable for use with a portable data analysis device 900, can perform malachite green detection directly at an aquaculture farm, and immediately transmits a detection signal to the data analysis instrument. Analyze with computer software and quickly learn whether aquaculture seafood or farm water contains malachite green. In this embodiment, the data analysis device 900 is composed of a signal processor (HP 34401A) 901 and an analysis device 902. The malachite green detector 2 signal is connected to the signal processor 901 for signal processing. Such as filtering, zooming in, and The signal is converted into a common signal processing such as a potential signal output, and the processed signal is transmitted to the analyzing device 902 for signal analysis to determine whether malachite green is detected. However, when implemented, the data analyzing device 900 does not have the above combination type. Limited.

The malachite green detector 2 includes a sensing electrode 3, a reference electrode 4, and a signal amplifying module 5 electrically connected between the sensing electrode 3 and the reference electrode 4.

The sensing electrode 3 includes a substrate 31 of an insulating material, a sensing film 32 coated on the surface of the substrate 31, and a conductive line 33 electrically connected to the sensing film 32 at one end thereof and extending outward from the substrate 31. And a package 34 fixed to the substrate 31 and the sensing film 32 and fixing the conductive line 33 to the substrate 31, the package 34 having a window for partially exposing the sensing film 32 340, the aperture 340 has a size of 3 x 3 mm ² .

The sensing film 32 has a conductive layer 321 which is fixed on the surface of the substrate 31 and electrically connected to the conductive line 33, and a butyl choline which is coated and fixed on the surface of the conductive layer 321 and exposed at the window 340. The esterase reaction layer 322, the butylated choline esterase reaction layer 322 can react with the malachite green to generate a signal, and the electrical signal is transmitted to the conductive line 33 via the conductive layer 321 for output.

In this embodiment, the substrate 31 is a PET (Polyethylene terephthalate) plastic sheet which is elastically bendable, but is not limited thereto. The conductive layer 321 is a hydrogen ion selective film. In this embodiment, ITO (Indium-Tin Oxide) is deposited on the surface of the substrate 31 by sputtering using DC magnetron sputtering (D.C. sputtering). The manner in which the conductive layer 321 is coated and fixed on the substrate 31 is not limited thereto, and the conductive layer 321 is not limited to indium tin oxide, and may be selected from indium tin oxide, tin oxide, zinc oxide, A group consisting of a mixture of cerium oxide and indium tin oxide, tin oxide, zinc oxide, and cerium oxide. The conductive line 33 is adhered to the conductive layer 321 through a silver paste and is fixed to the substrate 31. The encapsulating material 34 is an epoxy resin, and the substrate 31 and the conductive layer 321 are encapsulated by using good insulating properties and thermal coagulation properties, and only the butyric acid choline ester of the sensing film 32 is obtained. The enzyme reaction layer 322 is exposed to the window 340 while the conductive line 33 is fixed to the substrate 31.

The method for coating and fixing the butyl cholinesterase reaction layer 322 is as follows: the succinylcholine esterase used in the present embodiment is human acetylcholinesterase, but other butyl choline may be used instead. Esterase substitution. The human succinylcholine esterase source used was Sino Biological 12010-H08 Human BCHE.

The succinylcholine esterase was dissolved in a 10 mM phosphate buffer solution of pH 8 to prepare a butyl cholinesterase solution. After the conductive layer 321 is completely coated with the sputtering, and the conductive line 33 and the package 34 are disposed, 4 μl of the butyl choline esterase solution is dropped into the window 340, and moved to an environment of 4 ° C. The butyl cholinesterase was attached to the conductive layer 321 in an hour. Next, the same volume (4 μl) of 1% glutaraldehyde (GA) was added to the window 340, and then allowed to stand at 4 ° C for one hour to cause an interaction reaction between the butan choline esterase and strengthen The ability of the choline choline esterase to attach to the conductive layer 321 to immobilize the succinylcholine esterase to the conductive layer 321.

Finally, the sensing electrode 3 is placed in a 10 mM phosphate buffer solution of pH 8 and washed at 120 rpm for 5 minutes to remove the conjugated cholinesterase that is not immobilized on the conductive layer 321 or complete. The preparation of the butylcholinesterase reaction layer 322.

In this embodiment, the reference electrode 4 is a silver/silver chloride electrode (Ag/AgCl), but in practice, the reference electrode 4 can also be a conductive metal electrode such as gold, platinum, silver or copper, or other It is replaced by an electrode made of a conductive material, such as a graphite electrode.

In this embodiment, the signal amplifying module 5 is mainly composed of an instrumentation amplifier, and has an inverting input terminal 52, a non-inverting input terminal 51, and a signal output terminal 53. The sensing is performed. The electrode 3 and the reference electrode 4 are electrically connected to the non-inverting input terminal 51 and the inverting input terminal 52 respectively, and can be used to amplify and output the electrical signal generated by the sensing electrode 3. It should be noted that, in the implementation of the embodiment, the signal amplifying module 5 has circuit components, such as a power supply circuit, which are often used in combination with other amplifier circuits in addition to the instrumentation amplifier component, since the circuit components are The prior art is not the focus of the present invention and therefore will not be described in detail.

Because the instrumentation amplifier is mainly composed of three operational amplifiers (Operational Amplifier), and to increase its input impedance, the two operational amplifiers used as the inverting input terminal 52 and the non-inverting input terminal 51 are all field-effect transistors. The gate of the crystal (FET) acts as its signal input. Therefore, when the sensing electrode 3 is electrically connected to the signal amplifying module 5 After that, an extended gate field effect transistor (EGFET) structure can be constructed, so the present invention mainly sets the Ding on the extended gate sensing field effect transistor. The cholinesterase reaction layer 322 is used to detect the malachite green, and the signal amplification module 5 is used to perform signal amplification processing.

However, when implemented, the signal amplifying module 5 is not limited to the above-mentioned instrumentation amplifier, and may be directly replaced by a single operational amplifier, and the sensing electrode 3 and the reference electrode 4 are electrically connected to the non-inverting of the operational amplifier, respectively. The input terminal and the inverting input terminal can also construct an extended gate sensing field effect transistor structure, and can perform signal amplification processing.

The following describes the manner and effect of detecting the malachite green by the malachite green detector 2 of the present invention, so as to understand the detection limit of the malachite green detector 2. In order to compare the detection efficiency of the present invention with the existing malachite green detection method, the malachite green detector 2 of the present invention is used as an experimental group, and according to the manner in which the sensing electrode 3 is fabricated, the sensing film 32 of the sensing membrane 32 is used. The alkali esterase reaction layer 322 is replaced with an acetylcholinesterase reaction layer currently used for detecting malachite green, and then the sensing electrode 3 to which the acetylcholinesterase reaction layer is immobilized is electrically connected to the signal amplifying module 5 Take as a control group. The detection limit is defined as the lowest concentration of the analyte content when the reaction signal using the detection method is distinguishable from the background signal.

In the present example, the blank sample used in the experimental group was a 10 mM, pH 8 phosphate buffer solution containing 10 mM succinylcholine, and the blank sample used for the control group was 10 mM, pH 8 phosphate containing 10 mM acetylcholine. Buffer solution, and add different concentrations to the two blank samples The malachite green is made into a plurality of specimens 800 each having a different concentration of malachite green. The concentration of acetylcholinesterase on the sensing electrode 3 used in the above experimental group was 0.73 mg/ml/, and the concentration of acetylcholinesterase on the sensing electrode used in the control group was also 0.73 mg/ml. In this embodiment, after detecting by a plurality of malachite green sensors, the average value of the detection results of each malachite green sensor is taken, n=5.

When detecting whether the sample 800 contains malachite green, the signal output end 53 of the signal amplifying module 5 is connected to the data analyzing device 900, and the sensing electrode 3 and the reference electrode 4 are simultaneously Soaking in a blank sample, measuring a reference signal, and then immersing the sensing electrode 3 and the reference electrode 4 in the sample 800, so that the test group of the succinylcholine esterase reaction layer 322 and The succinylcholine and the malachite green in the sample 800 react to generate a sensing signal, and react the acetylcholinesterase reaction layer of the control group with the acetylcholine and the malachite green in the sample 800 to produce a The sensing signal is further analyzed by the data analysis device 900 according to the measured reference signal and the sensing signal change of the sample 800 of the blank sample and the different malachite green concentration, for example, the potential signal change, and the sample 800 of the experimental group is analyzed. The inhibition ratio of cholinesterase activity inhibited by malachite green, and the inhibition ratio of acetylcholinesterase activity of sample 800 in the control group, and the lowest detection of malachite green in the experimental group and the control group. limit.

Please refer to Figure 4 and Figure 5. From the control group, the concentration of malachite green and the inhibition ratio of acetylcholinesterase activity can be seen that the minimum detection limit of the control group is 1 ppm, and at the concentration of 1 ppm malachite green, 5% activity inhibition can be measured. Proportion, when the concentration of malachite green is less than 1ppm, the measured electrical signal cannot be The electrical signal of the blank sample is distinguished. However, the minimum detection limit of the experimental group can reach 10 ppb, and when the concentration of malachite green is 10 ppb, the activity inhibition ratio can be measured by 2%, and the detection sensitivity of the malachite green detector 2 made by the scorpion cholinesterase of the present invention is confirmed. The sensitivity of the malachite green detector made by the existing acetylcholinesterase is up to 100 times.

In summary, the sensing electrode 3 design of the butadiene cholinesterase reaction layer 322 is fixed through the conductive layer 321 , and the sensing electrode 3 is electrically connected to a signal amplification formed by an instrumentation amplifier or an operational amplifier. The non-inverting input terminal 51 of the module 5 constitutes an extended gate sensing field effect transistor structure, which can be used to reduce the volume of the entire malachite green detector 2 and is more convenient to carry and use. It has a good detection sensitivity of malachite green, and its minimum detection limit is up to 10 ppb. It is 100 times more than the detector made of acetylcholinesterase, which can more accurately detect whether aquatic seafood has a malachite green. Extremely high business value. Therefore, the object of the present invention can be achieved.

The above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, that is, the simple equivalent changes and modifications made by the patent application scope and patent specification content of the present invention, All remain within the scope of the invention patent.

3‧‧‧Sensing electrode

31‧‧‧Substrate

32‧‧‧Sensing film

321‧‧‧ Conductive layer

322‧‧‧ 醯 cholinesterase reaction layer

33‧‧‧Flexible wire

34‧‧‧Package

340‧‧‧ window hole

Claims (7)

A malachite green detector for detecting malachite green, comprising: a sensing electrode, comprising: an insulating substrate, a sensing film coated on the surface of the substrate, and a substrate fixed to the substrate and electrically connected thereto Detecting a conductive line of the film, the sensing film includes a conductive layer coated on the surface of the substrate and electrically connected to the conductive line, and a cover is fixed on the surface of the conductive layer and reacts with the malachite green to generate a signal A cholinesterase reaction layer. The malachite green detector according to claim 1, wherein the sensing electrode further comprises a package material that is coated and fixed on the substrate and the conductive layer, and the conductive wire is coated and fixed on the substrate, and the The encapsulating material has a window for partially exposing the conductive layer, and the butadiene choline esterase reaction layer is coated and fixed on a surface of the conductive layer exposed at the window. The malachite green detector according to claim 1, wherein the substrate is a flexible plastic plate. The malachite green detector of claim 1, wherein the conductive layer is a hydrogen ion selective membrane. The malachite green detector according to claim 4, wherein the conductive layer is selected from the group consisting of indium tin oxide, tin oxide, zinc oxide, cerium oxide, and indium tin oxide, tin oxide, zinc oxide, and cerium oxide. The group that makes up. The malachite green detector according to claim 1, further comprising a reference electrode, and a signal amplifying module electrically connected between the conductive line and the reference electrode, the signal amplifying module comprising at least one operational amplifier, and having An inverting input, a non-inverting input, and a signal output, the reference The electrodes and the conductive lines are electrically connected to the inverting input and the non-inverting input, respectively. The malachite green detector according to claim 6, wherein the signal amplifying module is an instrumentation amplifier.