TWI223070B - Method of detecting virus in a test solution using a quartz crystal micro-balance - Google Patents

Abstract

This invention discloses a method of detecting virus in a test solution using a quartz crystal micro-balance, which has following steps of (1) pasting a corresponding antibody of the to-be-detected virus onto the electrode probe of the quartz crystal micro-balance and, after being dried, measuring the frequency value f0 of the quartz crystal in vacuum or air to be set as a reference value; (2) placing the probe treated by step (1) into a solution containing no virus but same as the to-be-tested solution to measure the frequency value f1 of the quartz crystal in a no virus condition, which is used to calculate a change value between the frequency value f1 and the reference value f0, Deltaf1=f1-f0; (3) placing the probe treated by step (1) into the to-be-tested solution to measure the frequency value fm of the quartz crystal in the test solution, which is used to calculate a change value between the frequency value fm and the reference value f0, Deltafm=fm-f0; (4) comparing the frequency change value of step (2) to that of step (3), in which if the two values are the same, Deltaf1=Deltafm, this demonstrates that there is no virus in the test solution, and if the two values are different and absolute value of Deltafm is greater than that of Deltaf1, this demonstrates the existence of virus in the test solution. The invented method has high measurement sensitivity and low test cost and produces result fast, which can be extensively applied to virus detection of various liquid samples.

Description

1223070 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a quartz crystal microbalance for detecting viruses, and particularly to a method for detecting a patient using a quartz crystal microbalance in a test solution. Bone [Prior art] In the prior art, there are several methods for detecting viruses: 1. Molecular test (PCR) method: the direct detection of the sequence of the viral nucleic acid genome of a polymerase chain reaction. Tens of thousands, so this

The method verification process is time-consuming and costly. In addition, because the virus gene sequence of the same type is more repetitive, the test sensitivity is poor. 2. Immunofluorescence analysis (Immunofluorescence): use antibodies in serum containing virus antibodies as test reagents and mix the serum of patients with suspected disease. Combining and changing the fluorescence spectrum of the test reagent. With this method, the test result can only be obtained after there is a significant spectral effect, so it can be measured several days after the patient is sick, and the sensitivity and specificity are insufficient. Cell proliferation culture method: the virus isolated from patient specimens, plus

The method of reproduction 'can only provide evidence of a live virus, and the inspection process is time consuming. 4. The old method of quartz crystal microbalance: At present, the antibody is placed on the electrode probe of the quartz crystal chip at least, the antibody and the corresponding virus are combined to produce a trace mass increase, and then the -π law (Schubert's law is as follows No), the measured frequency change is derived from the easy-to-measure frequency change, which leads to a slight increase in mass. Δί = — 2f02 *] Διη 5 1223070 where: △ f: frequency change, △ m: mass change, f0: original frequency of quartz crystal, P q: quartz density,

Cq: coefficient of deformation of quartz; plus solid mass of 1 for use =: or quartz crystals in the air "^ Th. V Thickness used in vacuum evaporation for monitoring

= Almost all test liquids such as blood, saliva and other secondary toxic liquids are tested, which is what the present invention is to study _ Thousand Pairs of Disease [Summary of the Invention] The purpose of the present invention is to provide _ species that can be tested; Method for detecting virus using quartz crystal microbalance. To achieve the above objective, the present invention adopts the following technical solutions:

Quartz crystal microbalance is used in the test solution to test the toxic time. The steps are as follows: ⑴ Adhere the antibody corresponding to the pre-detected virus to the probe of the quartz crystal microbalance, and dry it to measure the frequency of the quartz crystal in vacuum or air. f〇 'and use it as a reference value; (2) Put the probe treated in step 人 into a human without virus, but in the same solution as the fish to be tested, and measure the frequency h of the quartz crystal in the absence of virus in the solution. Calculate the change between the frequency value and the reference value f f; 6 1223070 (3) Put the probe processed in step (1) into the test solution, and measure the frequency of the quartz crystal in the test solution fm, calculate the change between the frequency value fm and the reference value △ fm = fm-f〇; (4) Compare the frequency change values of step (2) and step (3), if the two values are equal, the test solution No virus; if △ ^ and ^ fl have different values, △△ fmj > | Afi 丨 proves that there is virus in the test solution. The frequency change value △ fi calculated in step (2) should conform to the law (Kanagan's mutual law):

… [One plant] ^ qCq V wind type · △ fl · Shiyang crystal puts in the solution to produce the frequency change amount, f0: original frequency of quartz crystal, P q: density of quartz crystal,

Cq · Quartz deformation coefficient, P 1: liquid density, π 1: liquid viscosity.

The frequency change value Aim calculated in step (3) should conform to Martin's law (Martin's law): 2f〇2

[P

Pi Πι where: Δίπι ·· frequency change of thickness and mass change in solution fo ·· the original frequency of quartz crystal, 7 1223070 q: density of quartz crystal,

Cq · quartz deformation coefficient,

Ps · Density per unit area of the mass layer with increased surface thickness, P 1: liquid density, β 1: liquid viscosity. When comparing the frequency change values of step (2) and step (3), if the frequency change of the quartz crystal in the test solution conforms to Kanazawa's law, it is proved that there is no virus in the test solution. If the frequency change of the quartz crystal in the test solution conforms to Martin's law, That is, 丨 μ 丨 > 丨 _, it proves that the test solution: virus exists, and the micro mass of the virus antibody and antigen binding on the quartz crystal microbalance probe can be calculated by Martin's law. A plurality of quartz crystal arrays may be provided on the quartz crystal microbalance, an electrode probe is provided on each of the arrays, and

Corresponding antibodies of different viruses are attached to the needles to detect multiple viruses at the same time. T The present invention has the following advantages due to the adoption of the above technical solutions: 1. The method of the present invention uses the principle of combining disease, antibody on the quartz crystal microbalance probe with the virus antigen in the test solution, and : The frequency of quartz crystal in air or vacuum, measuring the frequency change of quartz crystal in virus-free solution, and measuring the frequency change of quartz crystal in the liquid to be tested; and comparing the frequency change to achieve the quartz crystal microbalance in Purpose of detecting virus in liquid. 2. The present invention introduces Kanazawa's law and Martin's law, which not only draws conclusions about the presence or absence of virus, but also can accurately measure the amount of virus, and its sensitivity can reach <10.8 g. Goods 8 1223070 3. The present invention not only proposes a method for measuring a virus using a quartz crystal microbalance in a liquid, but also the method can detect compared with the existing measurement methods. Convenient, fast results, low detection cost, especially in A plurality of quartz crystal arrays are set on the quartz crystal microbalance, and the individual antibodies corresponding to different viruses are adhered to different quartz crystal probes, which can simultaneously detect multiple trace viruses in the test solution at the same time, and can also detect duplicates. cross-infection. This is currently the world's most sensitive method for detecting virus in test solutions. The method of the invention can be widely applied to the virus detection of various liquid samples. [Embodiment] This case will be described by the following examples to describe the spring technical features of this case in more detail, and to confirm the uniqueness and feasibility of this case. Example 1: Detection of herpes virus (human herpes Viruses) quartz crystal microbalance using AT cut circular quartz crystal wafer, diameter 8.0mm, circular metal electrode diameter 4.0mm, thickness 200nm, plane fundamental wave, 10MHz quartz crystal probe, first immersed in CMV65_kDe antibody solution, so that a single layer of CMV65-kDe antibody adheres to the surface of the quartz crystal probe, after drying, measure the original frequency f () of the quartz crystal, and then put the probe needle There is no disease, but in the same solution as the test solution, measure the frequency value of the quartz crystal in the absence of virus in the solution. According to Kanazawas' law, it can be seen that the frequency value A is reduced, and the frequency value is equal to the reference value 4 The change value between △ [=. Immerse this quartz crystal probe with CMV65-kDe antibody in a test solution that may contain herpes virus. At this time, if herpes virus is present, the antibody will combine with the antigen of herpes virus to detect the quartz crystal. Needle surface 9 1223070 The thickness (mass) increases, which conforms to Martin's law, and the frequency of quartz crystals is further reduced, that is, | Aiml &gt; 丨 Afil, you can detect the presence of effervescent virus in the test solution, and complete the test. If the measured value of the frequency change of the quartz crystal △ fm = Z \ fi is in accordance with Kanazawa's law, it means that there is no effervescent virus in the test solution, and the inspection is completed. Example 2: Detecting antihuman immuodefficiency viruses (HIV) Quartz crystal microbalance uses AT to cut a round quartz crystal wafer, with a diameter of 8.0mm, a diameter of a circular metal electrode of 4.0mm, a thickness of 200nm, a plane fundamental wave, 10MHz Quartz crystal probe, first immersed in anti-HIV manoclonel antibody solution, so that a single layer of anti_HIV manoclonel antibody adheres to the surface of the quartz crystal probe, after drying, measure the original frequency fG of the quartz crystal, and then put the probe into Virus, but in the same solution as the test solution, measure the frequency value fi of the quartz crystal at this time. According to Kanazawa's law, it can be seen that the frequency value fi decreases, and the change value between the frequency value fi and the reference value fG is fl-f0. The quartz crystal probe with the anti-HIV manoclonel antibody attached is immersed in a test solution that may contain an immune virus. At this time, if the immune virus is present, the antibody will be combined with the antigen of the immune virus to make the quartz crystal probe As the thickness of the surface increases, it conforms to Martin's law, and the frequency is further reduced, that is, | Aiml &gt; 丨 Δί \ |, then the presence of immune virus in the test solution can be detected, and the test can be completed. If the measured frequency change of the quartz crystal is in accordance with Kanazawa's law, it indicates that there is no immunodeficiency virus in the test solution. 10 1223070 The test is completed. Example 3: Simultaneous detection of multiple viruses with multiple quartz cells; The computer can arrange and use the probes at the same time, and detect the corresponding individual antibodies of different diseases in different quartz crystals. Π1 4 + ^ to read out each quartz crystal body at the same time. Quartz crystals used in crystal microbalances include various incisions

And other cutting methods, such as at, bt, ct, dt, fc, and scales to detect disease weight-the formula described above: to provide a method to use quartz crystal microvirus in the test solution: two can be used to solve the conventional quartz crystal micro Accuracy of balance detection, shortening of B 2 ::: shortcomings of body substances, and can effectively increase = 逑 The examples are described in detail and can be modified by those familiar with the art 2 顿 and modified for the sake of ㈣㈣ 如 ㈣ Scope protection

11 1223070 [Schematic description]

Claims (1)

^ 223070 The scope of patent application: 1 · A micro-step using quartz crystals in the test solution is as follows: The method for detecting viruses by a balance, its toxicity ^) = The electrode probes of quartz crystal micro-balances adhere to the antibodies that should be used for pre-detection of sick patients After drying, measure the quartz crystal in vacuum or air frequency value f0, and use it as the reference value; 〃, ⑺ Put the probe after step ⑴ into No Disease #, make a fish to be tested, and measure the solution In the absence of a virus, the value of the quartz crystal, the value of the out-of-rate value ^ and the reference value f0 is a value fVf0; (3) Put the probe processed in step (1) into the test solution, measure the frequency value fm of the quartz crystal in the test solution, and calculate the change between the frequency value &amp; the discontinuity value f0. (4) Compare the frequency change values of step (2) and step (3). If the two values are equal, it means that there is no virus in the test solution; if the value of △ ^ is different from the value of 8 and | 仏 | N Afi | proves the presence of &quot; virus in the test solution. 2. The method for detecting a virus using a quartz crystal microbalance in a test solution as described in item 1 of the scope of patent application, wherein the frequency change value Δ fi calculated in step (2) should conform to Kanazawa's law: Afi = [-2f 〇2 Pi ni VP qCq 4nf0 In the formula: △: Quartz crystal is put into the solution to generate frequency change, f〇: Quartz crystal original frequency, Pq: Quartz crystal density, Cq: Quartz deformation coefficient, pi: Liquid density, 77 i: liquid viscosity. 13 1223070 days; 4 The method of using quartz crystals in the test solution as described in item 1 of Erjiwei, where the frequency value △ fm calculated in step (3) should conform to Martin's law: 2f〇2 Pi n \ ~] P qCq 4Tif0 thick, frequency change from two to two, f »Kai M private / Pq 疋 quartz crystal density, &amp; is quartz change ?, two ~ 疋 increase the surface thickness of the mass layer per unit area density , D 1 · Liquid density, 々1 ·· Liquid viscosity. When the frequency of the quartz crystal in the test solution is changed to Kanazawa's law, &gt; T is determined by the presence of poison in the test solution, pass = increased trace mass. Please bind the disease-free antibody to the antigen. The method for detecting a virus in the balance described in the above item is characterized in that a plurality of quartz crystals are provided on the receiving surface of the crystal, and a micro-electrode probe is arranged on each of the quartz crystals. Simultaneous detection of multiple in the test solution: two different virus phases 14 izzju / υ 6 · — A quartz crystal microbalance was picked up. The method steps are as follows. It is applied after the test solution is dried (ΥΓΛ crystal microbalance). An antibody is adhered to the electrode probe, and it is dried! The frequency value of the quartz crystal is measured under the gas state as a reference value f0; Identical two ', the first frequency value of the quartz crystal when the first frequency value of the quartz crystal in the absence of virus in the volume solution is out of the first frequency value f] and the reference value f. The change value △ [=; In the test solution, measure the second frequency of the central crystal of the Shiyang crystal in the test solution + 瞀 + 进 山 进 ^ The second change between the second frequency value and the base value f0 and (4) compare the The first and second frequency changes, when ^ y △ fll There is a virus. = The method using the quartz crystal microbalance detection method described in item 6 of the patent application, the mother method, wherein the antibody corresponds to the pre-detection virus. 8 The use of quartz as described in the item 6 of the main application patent scope A method for detecting a crystal microbalance, wherein the first-frequency change value M should conform to the law of Hall H: Af, = [-2f〇2 Ρι Πι 4 ^ f0 • In the formula. ΔΑ: The frequency of a quartz crystal in a solution generates a frequency Amount of change, f0: original frequency of quartz crystal 'Pq: quartz crystal density, quartz deformation factor 15, ρ liquid density' π 1: liquid viscosity. 9 · If the patent application Fan Tianping detects the square half of the virus, the term In the test solution, the quartz crystal micro-Martin law is used, where the second frequency change value Δ fm should conform to Is the frequency change of the thickness and mass change in the stone body ϋϋΓ, f-shaped coefficient,] There is a rate, Pq is the density of quartz crystal, Cq is the change of quartz • 疋 Increase the unit density of the surface thickness mass layer, Liquid density, ^ 丨: Liquid viscosity. • The method of detecting the disease using a quartz crystal microbalance as described in item 6 of the main patent scope, when comparing the frequency change value of step (2) and step (3) 4 'If the frequency of the quartz crystal in the test solution The change conforms to the &amp; bribe law, and Δfrn ^ Afi proves that there is no virus in the test solution. U · As described in Item 10 of the scope of the patent application, a method for detecting disease using a quartz crystal microbalance. If the frequency change of the quartz crystal in the test solution conforms to rtin's law, and 丨 仏 丨 & 丨 Afi 丨, then It is proved that the test solution has disease = existence, and the micro mass of the virus-antigen binding on the needle collected by the quartz crystal microbalance can be calculated by Martin's law. 12. The method for detecting virus residues by using a quartz crystal microbalance as described in item 6 of the scope of the patent application, wherein the quartz crystal microbalance is provided with a plurality of = central arrays and is set on each of the central crystals. The electrode probe is set to 16 1223070 needles, and the corresponding antibodies of different viruses are attached to each electrode probe to detect multiple viruses in the test solution at the same time. 17 "Invention Patent Specification 7 (This manual is in the format, order and bold type, please do not change it arbitrarily, ※ please do not fill in the mark) ※ Application number &quot; ...: Application date classification such as Ryume &gt; I. Invention Name: (Chinese / English)) Method for detecting virus using quartz crystal boat microbalance in test solution. Author, applicant: (total) Name or name: (Chinese / English) 1 · Hou Bangwei / p〇nfe i Hou Representative: (Chinese / English) Address of residence or business office: (Chinese / English) Hsin An Road, Hsin Chu Science-Based Industrial Park, 3rd Floor, 8 Hsin On Road, Hsinchu Science Park, Taiwan R. 0. C. Nationality : (Chinese / English) ROC Participant and Inventor of the Republic of China: (Total 1A) Name: (Chinese / English) Hou Bangwei / P〇nWei Hou Residence or Business Office Address: (Chinese / English) Hsinchu Science Park Hsin-an Road S ^ n An Road, Hsin Chu Science-Based Industrial Park, Taiwan R. 0. C. Nationality: (Chinese / English) Republic of China R.〇.C