TW200528179A - High sensitive multi-channel air-virus-counting device and method - Google Patents

Abstract

The invention disclosed a high sensitive multi-channel air-virus-counting device and method. The device is using Micro electro mechanical System (MEMS) technology to manufacture multiple particular and independent channels, each channel include electrostatic precipitation devices, separation devices which employ electrophoresis and dielectro force and a nano-pore featured as ion-channels. The electrostatic precipitation devices employ static electricity to trap virus in air, every nano-pore consists with a nano-ampere order measuring circuit that can measure ion-current, an ion-channel electric field that can manipulate the virus, and a controller for coordinating the whole system. Vacuum absorb fixed quantity of sample air through electrostatic precipitation region, let virus through air/liquid interface into channel with solution and flowing in sample solution collected trough, then use DEP force or electrophoresis that produced by separation device on the channel to induce and separate viruses. Next, move the separated particular viruses to nano-pore. When the virus pass through the nano-pore one by one, the nano-pore is blocked and results in the decreasing of ion current. Recording the numbers of ion current reduction events can correctly count the quantity of viruses in the fixed volume of sample solution. For different virus a different separation electro-field pattern can be employed accordingly, therefore each independent channel can synchronously proceed the counting of same or different virus, the disclosed technology can achieve the purpose of air-virus count.

Description

200528179

V. Description of the invention (1) [Technical field to which the invention belongs] A multi-channel, high-sensitivity air station is used to accurately calculate the quantitative empty $ = virus count (detection) split refers to the quantitative Air test 5 = the number of viruses ^ the virus in the test specimens and counted: j = import, automatic separation [previous technique]

In recent years, the health hazards of biogas rubber have gradually been affected by subtropical regions, and the climate is warm and humid. Above Taiwan ^, the average temperature is between 2. . Bu 3 (^ of: phase = degree average growth at 70%, You Dou in this guest your firefly 悴 悴 10 knife suitable for microorganisms containing i because of the existence of a relatively high concentration of biotin hair "bio aerosol environment Debt measurement technology to obtain a representative sample is a very important subject. Biogas gel refers to biological particles suspended in the air, which come from a wide range of sources, including: viruses, bacteria, radioactive bacteria (8 (^ 111 〇1115 ^ 46), fungal, moss, and fern (fer η) spores

(algal) and plant cells; insects and their fragments and excreta; proteins from animals and plants; enzymes, antibiotics, and other products made by biotechnology; endotoxins from Gram-negative bacteria; Mycotoxin and glucan. There is currently no effective medicine to treat and prevent viral disease. The only effective way is to isolate the virus from invasion. Past research has selected three types of impact bottles, membrane filter paper and pectin filter paper.

Page 11 200528179 V. Description of the invention (2) Biogas adhesive connector used (sampling flow rate, room evaluation, discussion of physical factors (the biogas adhesive itself can withstand 23 gan sampling time) and biological sampling efficiency (inlet Efficiency 隹 丨) The physical recovery rate of the bacteria it collects: colony survival efficiency and relative survival rate-like efficiency (total 〇 Evaluation of Environmental Health Group Hao Meizhi ^ Stomach Reference Bench-like Technology) Large = resistant; 2 strong and weak is to determine the biological sampling efficiency of the sampler. When the E. coli is collected, it will be washed at the door ^ Yitian Qian & Xiang Dangluo survival efficiency is about 5%), ^ ^ 'The rate is the best (its bacteria are poor; and the recovery rate of the impact bottle and thinner, two ,, and is about that of the impact bottle. When collecting Escherichia coli with 4-membrane filter paper, tart is a factor, #cup卢] R 八 # 胜 枰 国 ^ This time is not large. It is best to be within Η, the impact of the sampling flow is close when collecting B. subtilis, but the recovery rate of thin paper is about σ right = fruit The recovery rate of adhesive paper is about the same, and it is not affected by 70% of the rubber paper is produced in the low-concentration product, the sampling time, the amount of the machine, and the sampling time. In the low-temperature latex environment, the sampling time =: The situation where it is not available. J + And it is short, otherwise the above method of collecting bio aerosol does not apply to the material. The collection time on the material is too long, so it is not right: :: The mechanism for miniaturization is to add sampling and collect. Another U: It is not easy to use it effectively in ordinary environment. It is rarely mentioned on the spot. The technology of aerosol virus collection is very similar to that of Mao Maoyue. It specially proposes a miniaturized electrostatic sinking jealousy to change the collection effect and promote it to be handheld. Can be easily installed

Page 12 Previous units of concentrated microparticles, mixed particle number, indirect counting assay) Red blood cell knots and a series of hemaggul tears 79 On-site sampling collection and instant analysis in general environments, and can also be treated with airborne viruses. Do you generally use aggregation for virus detection methods? Linkage (PCR) detection technology. Use specific aggregations? Chain Should primers be polymerized according to the DNA of each virus? Consistent genes are designed to generate relative base acid fragments only when the virus-detecting gene is used as a template. The specificity of the primers to the virus is used to detect whether the virus is susceptible. However, the PCR detection method is time-consuming and requires The experimental officer can be prepared by the company organization or the family by itself. V: It is used to the expense: More than three. For this reason, the present invention [virus counting device] allows a general company organization to use a self-provided inspection device. A quick and low-cost test of the quality of the air is whether it is severely infected, mildly infected, or non-infected (that is, the number of viruses in a single accumulation of air is more recruited), and effective treatment is immediately taken to prevent injuries caused by time delay. "

The methods for counting viruses include direct counting and indirect measurement of disease. The method of direct counting is to compare the number of viruses and known concentrations under an electron microscope and calculate the number of viruses I. The counting result and the concentration of latex particles are converted to the concentration of virus Z. The most common method is hemagglutination. When the number of viruses and red blood cells When the ratio is large enough, the virus will form a network, causing the suspension to precipitate or agglutinate. Mixing the virus dilutions will cause the highest dilution of blood clotting to occur in the titer. This method can be accurately and quickly determined. Infected, and high or around the home, the infection with latex and microt and blood cells is a poison 200528179 V. Description of the invention (4) Number. The end method (end ρ 〇intmeth 〇d) is a series of dilution of the virus suspension Then, inoculate the animals, and draw the curve of the animal lethality caused by each dilution to find the endpoint dilution that caused 50% of the animal's death. LD50 (Lethal dose) and ID50 (Infectious dose) refer to here The dilution contains sufficient doses, which can cause 50% of the animals to be lethal or infected. Plaque assay is to dilute different dilutions Several phages or animal viruses are mixed with the host cells and spread on the surface of the plate. When the virus particles cause infection on a large area of host cells, the cells will be lysed to form bacterial plaques. Each plaque is caused by a virus particle. As a result, calculate the number of plaques and multiply by the dilution factor to get the concentration of the original virus infection unit. In nW0 02/052246 A2 Method and Device for

Detecting and Identifying Bio-aerosol particles in the airn ATOFMS (aerosol time-of-flight mass spectrometer) in the particle flow, selected using glory technology 'and using MALDI (matrix-assisted laser desorpt ion / ionizat ion ) The selected aerosol particles are ionized. After that, the formed ions are detected, so the aerosol particles can be identified. However, this method requires extremely expensive equipment. It is known from the above methods that the number of viruses needs to be calculated, some are diluted, some are observed with a microscope, and some require expensive equipment. These processes will only make the process more complicated and have more variables.

200528179 V. Description of the invention (5) The device made by an invention does not only need a cumbersome pCR process, but also does not need to use a manual method to observe the amount of virus. The invention only needs to introduce the specimen into the device, that is, y will accurately The virus figured it out. Compared with the previous technology, Ben Maoyue is easy to operate. Generally, people without party training can use it, and the accuracy of observation is much higher, and the price is cheaper. [Content] The amount of the present invention, the visual inspection of the present invention is generally done in the tedious objective of the present invention, the user of the present invention issues a warning, the objective of the present invention, using half of the objective of the present invention For the purpose of visual inspection of the present invention in the future, one of the two different types of prevention of the company ’s two company ’s testers, three of which are not required, four of which are immediately collected, five of the five-conductor analysis, and seven of the viruses

’It can be quickly concluded that the airborne virus cluster in the environment is infected with the virus. Improve the existing virus detection technology, so that virus detection or home can do it, without having to get a laboratory method. , From sampling to detection and counting to interpretation, one can complete the operation of biochemical knowledge. Run to identify if there is any danger of virus infection and take effective measures.

The r Γ plant can be integrated into the chip loading and loading, and the micro-electromechanical process to complete. track. Store in any portable media, using Crystal M μ ,. The independent flow channel of the eve group can be detected simultaneously

Page 15 200528179 V. Description of the invention (6) Technical content Air suspension process 1. Sterile filtration of micro particles obtained from the specimen. Process 2. Sufficient charge in the air before circulation. The electric virus will move to the traction and contain the disease to make the virus. During the moving process, the disease enters the center of the electrode, and the electrode is in the area of the electrode pole. During the process, the disease will have the virus, and then the droplet is sealed. Counting the complete wafer to obtain sample 1 is to use a vacuum pump to fix the membrane to filter. After this step, obtain the constant volume treatment (charging and electrostatic precipitation) from the environment to be tested. Viruses can be used first if necessary, followed by electrostatic precipitation. In the switch, the poisoned liquid of the electrode is switched to the droplet encapsulation and the subsequent analysis and analysis device is separated. The first stage of the analysis device will pass a high-frequency alternating current, so that other parts that are not viruses will 'change the electric field. The slowly moving process shows that at a time, a constant flow rate of 5 // m passes through 5 // m to get rid of most of the dust and accumulated air; 2 The parent stream charges, making the disease Put on the method of “making the high electrostatic field adsorption band point” to make the electric field change the area that moves slowly to the pole, so as to automatically pass through the separation electrode, the poisoning on both sides is repelled by the dielectric force to the flow channel and it is adsorbed on the electrode, and the traction virus is switched to The solution of electroporation traction 4 of the droplet package is converted by the electrode, so that the droplet forms a droplet package, and the packet is sent to the area of the ion channel. After the separation 5, a small amount of virus will remain in the area of the separation electrode. For counting purposes, Can do secondary separation, when the virus passes the traction map

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Page 16 200528179 V. Description of the invention (7) Electrode formation droplets will be less separated after the first separation. The virus is separated from the four steps of the process. When the virus drops; the process of the current is to open the electrode first. After leaving the virus, the virus will pass through the second stage, and then it will be separated by the second stage and then separated again, and the electricity will be separated: ^ can be miscellaneous coins 1 Mouth 丨 For the second adsorption, she applied an electric field to the electrode. ^ ^ ^ ^ "Into the knife" according to the process of the third and the disease is not sent to the virus counting ion channel, · When counting 6 subchannels will make The channel section 1 ^ 7 < ° 丨 clogged, causing the number of secondary currents and the detection of the number of secondary poisons. The results were analyzed. II: Calculate the number of independent tests in multiple groups to achieve the goal of disease-free counting. Because the virus in the air collection device is mainly in the form of _ Jiao Yi Ruo # 用 一 本本> 丄 h micro-sized droplets, so the right side will use a filter of political water about a dream to vortex when it is a lotus root. , + + ~ Display, may cause the virus droplets to stay in the past / wish to be placed and cannot be effectively collected, see paper and fruit-like: extravagant. Another possible impact bottle, film for analysis F belt ”Shi Yi ,,,, and the movement of the virus to other tests are analyzed and displayed, so the present invention is particularly tart with lettuce and thunder π into ^ peach, and the method of electrostatic precipitation is used. Virus ^ ^ ^ / L Zn., Pure water of mother and master, so that the virus can be automatically moved to other inspection and analysis devices. For those who are not sufficiently charged with biogas gel, they can use AC electric field to charge

200528179 V. Description of the invention (8) Electric appliance (charger), references MLackowski, A. Jaworek, A. Krupa, " Current-voltage characteristics of alternating electric field charger ", J. of Electrostatics, 58 (2003) 77 -89, the generally applied frequency is about 50-400 Hz, and the electric field size is about lkV / cm-10kV / cm. Since the purpose of the present invention is to miniaturize the system, the electrode forming the electric field charge is at a distance from the car reference. The literature may be less than 50 Oum. In order to charge the biogas tape and not deviate to the electrode plate during charging, the present invention does not use DC charging, such as reference [1] Mainelis, Gediminas; Adhikari, Atin; W i1 1 eke, Klaus; Lee, Shu-An; Reponen, Tiina; Grinshpun, Sergey A. " Collection of airborne microorganisms by a new electrostatic precipitator "Journal of Aerosol Science Volume: 33, Issue: 10, October, 2002, pp. 1417-1432 And [2] Mainelis, Gediminas; Willeke, Klaus; Baron, Paul; Reponen, Tiina; Grinshpun, Sergey A .; " Electrical charges on airborne microorganism s " Journal of Aerosol Science Volume: 32, Issue: 9, September, 2001, pp · 1087-1110 o There are two main factors affecting the minimum voltage Vmin of Corona discharge: 1. Dielectric strength of air: too It is about 3 kV / mm. 2. The shape of the electrode: · The larger the curvature, or the smaller the radius of curvature R, the lower the Vm η and the higher the opposite.

Page 18, 200528179 V. Description of the invention (9) If a needle electrode is used for a flat plate (need 1 e ~ t. Η 1 ^, ^ ^ ^ ^ > Al, t0 —Plate), the corona discharge is set. And Lu, the electric field is the strongest near the needle electrode surface and p bucket surface:

Emax = V / R kV / mm XR = Vmin af, when Emax > 3 kV / mm, that is V > corona discharge will occur. ΊThis model estimates various radius of curvature❻ hlOOiim, Vmin = 30 0V; R = i〇r / mv. mvmin ^ h

Vuin ,. ㈣, Vl " in = 30V; R = 1, so with miniaturization and portable settings over 500 _, the applied voltage is two, two: the distance between the poles does not exceed this. Of course, microelectromechanical technology can be used to pin the tip diameter. It should be less than 3 _, produced by electrosurgery, and the other one uses an array of nano carbon f arrays to achieve this. The second reason is explained by using rice and reason. • The carbon tube plane shows: Camera powder and the hair " The electrons issued by the Ming / Slave officials can directly hit the biogas glue in the air of the anode plate, // The electrons emitted by the disgusting official can impact the attachment, " Tai Huang Rongtang, etc. The Republic of China invention patent application for the second electric μ electric device " Yuan / Guan 9 'application number increases the frequency of charging the second electric current to, and the test is based on basically, regardless of the static electricity ::, the distance between the two swings. Electricity / children's age, or AC charging, the movement behavior is based on page 19, 200528179

The following formula md (dx / dt) / dt mg 71 ^ rcx (u-dx / dt) where g is the acceleration of gravity, the amount of charge of the q particles, the viscosity of the m particles, and the viscosity of the cx drag. "" Mammary ^ "Figure 3" The virus-containing bio aerosol gas is introduced into the wafer inlet by the ^ acuum Pump), so that the gas flow rate reaches Libang-lm / sec, first through the AC charging area to generate, charge , And then through the electrostatic precipitation area, because the biogas glue% has a face, so under the action of the DC electric field / ° electricity: object = = tank to place liquid, preferably to: Erpu = iff in water The process of sampling is all done by vacuum pumping: 25 sets of sampling volume,-about 6 ° cm3, to prevent, island girl = can be installed with a filter below 5 microns at the inlet-side & Announcement ==, fire dust and other non-biological aerosol particles enter the water, and another biogas: "When Qi Bangpu is not in operation, the passing device can also reduce the hydrophobicity of the collected ^ surface ^ = diffusion of the liquid from the outside world communicate with. Because the downstream of the groove is for explanation, the machine path, and the mechanism of forming droplets (see the next section of the tour: after the next: Γ; the aqueous solution containing bio aerosol will be able to be transported to the lower tip ΪΓΐΐ Virus isolation and counting. It takes time for negative ions to strike the biogas gel to make it negatively charged and charge the office.

Page 20, 200528179 — V. Description of the invention (11) dq / dt = 3 7rn (t) // E (t) r * r * [lq (t) (er + 2) / 12 7Γ eOE (t) r * r * er] * [1 -q ⑴ (er + 2) / 12 7Γ εΟΕ ⑴ r * r * er] where r is the particle radius, and ε r is the relative permittivity of the particle, // Is ion mobility (m0bi 1 ity), n is the space electric charge, degree, and β is the electric field. Charged after infinite time ^ The saturated charge absorbed by the particle is

qs = 12 7Γ ε Or * r * Em * ε r / (ε r + 2) where Em is the amplitude of the AC electric field. Refer to Figure 4. Needle-shaped electrode to flat plate (needle — t〇_pUte) or targeted corona The discharge device can be manufactured by using MEMS process or array nano-carbon technology. The use of MEMS process is as follows. First, P_Type (100) material crystal stone is selected as the substrate.

Step 2: Use the photolithography etching technique to softly engrav the upper half of the substrate, and use the formula shown in Figure 4 below, and then place the substrate in 85 ′ potassium oxide (K0H). Tong Caiyi, Jia, ut 逭 raft edge etching, using the characteristics of single crystal silicon material to etch a 54.74 degree inclined groove, as shown in Figure 4 (c) " Step 3, using acetone to wash away the photoresist As shown in Figure 4 (d) Step 4, the method of making electroforming deposits a layer of Ni metal on the surface of the substrate,

Page 21 200528179 V. Description of the invention (12) Figure 4 (e) Step 5: Then place the base material together with the metal in (K0H), and engraving while mixing, and then oxidize the remaining oxide as shown in Figure 4 ( f) The substrate is etched away, and the photolithographic etching technology is used to ... on the metal layer (spacer), and finally, steps 1 to 5 are used to connect to the spacer to form a closed and warm space. '. The other—Ni gold using nano carbon control process is as follows: Step 2 Step 3 Step 4 Step 5 Use a photoresist coater to spin-coat a rhino on the substrate and use light for shadow #etching technology to reach the nano carbon tube. '^ The catalyst metal (Ni, coF shape) was deposited by an evaporation machine. The catalyst metal array was completed using the e-off technology. ^ The catalyst metal was annealed or reduced by using a high temperature furnace. And metal or silicide. ', Make eight into Nai step six, and then use carbon, hydrogen and other carbon tubes such as acetylene methane. ^ Homogeneous rolling phase to generate nanometer M h For cost considerations, you can also use screen printing to Nai米 舻 # It can be turned into an array shape, and then it can be processed at high temperature. A A & 'Virus separation device is shown in Figure 5. To achieve the purpose of separation, the first characteristic; · Born to use the dielectric power DEp to achieve the "unlimited" electrical characteristics, electrical conductivity needs to be known, in the case of / the virus dielectric constant

Page 22 200528179 V. Description of the invention (13)

In the mechanism, two methods can be used. One is ROT measurement, which can be referenced as M Changes in Friend murine er yt hr o 1 eukaem ia cell membranes during induced differentiation determined by electrorotation, f, Biochimica et Biophysica Acta 1193 (1994 ) 330-344, and Diferences in the AV electrodynamics of viable and non-v i ab1e yeast cells determined through combined dielectrophoresis and electrorotation studies ", Phys. Med. Biol., 1992, Vol. 37, No 7, 1499- 1517 · The other is to use Levitator to measure. References such as Integrated planar concentric ring dielectrophoretic (DEP) levitator ", Journal of Electrostatics 55 (2002) 65-79, use these two methods to find the cell permittivity to facilitate integration on the wafer. The frequency of the electric field to be provided for the separation. Requires a high degree of precision in the measurement, such as ... impurities in the air, room temperature, the length of the cell culture time, the characteristics of the medium solution (high osmotic pressure solution, low osmotic pressure solution and isotonic solution), Whether the electrodes are clean or not is enough to affect the measurement results.

Methods for using DEP to isolate viruses in the literature, such as " Separation of Submicron Bioparticles by Dielectrophoresis' 丨, Biophysical Journal Volume 77 July 1999 516-525 and f, D 1 1 ec t rophoretic tic Trapping of Dissociated Fetal Cortical Rat Neurons " , IEEE Trans, on Biomedical Engineering, Vol. 48, NO · 8, AUG 2001 921-930 and

Page 23 V. Description of Invention (14)

Trapping in AC Electrode cages, Journal 〇f

Some people have studied in Electrostatics 50 (20 0 0) 1 7-2 9 and other related literatures have mentioned that they found that different specimens have different dielectric properties and conductivity, so the present invention Based on this feature, the dielectric field is generated by applying an external electric field, and the principle that different dielectric frequencies and frequency changes between different specimens will cause a polarity change, and then the electrodes 2 3 are made by microelectromechanical process technology. An alternating current is added to the designed electrode to generate an electromagnetic effect, and then the frequency is changed so that different specimens will have different dielectric characteristics at different frequencies. Adding different positive and negative electric fields to the electrode will cause Samples with different polarities are distinguished to achieve the purpose of separation. The invention uses the staggered electrodes 24 to make use of the electric field formed by the external partial voltage circuit and makes the electric field drive the particles (including viruses) in a phase backward manner. First, the particles 21 to be tested are driven to the flow channel by the dielectric power. The center, and the other substances 22 in the specimen will be adsorbed on the electrode. Use particle traction device, and then use the method of backward phase, such as "", FF, Wang, Huang, Y., Pethig, R., Vykoukal, J., and Gascoyne, PRC. Proc. Natl. Acad. Sci ., USA · 92 ·· 860-864, 1 9 95, the particles 2i to be counted are concentrated in the center of the flow channel, so that they are drawn along the flow channel to the position of the particle (including virus) traction electrode 26, The area of the traction device is hydrophobic, so when the sample flows through the separation electrode to the traction electrode, the sample will stay on the right side of the hydrophobic line 2 7 due to the relationship of hydrophobicity. The electrode formation is used to pull the solution forming droplets to the next 200528179. V. Description of the invention (15) References for virus traction devices 丨 'Towards Digital Microfluidic Circuits: Creating, Transporting, Cutting and Merging Liquid Droplets by Electrowetting-based Actuation " IEEE MEMS 2002, mentioned that when a liquid is on a hydrophobic surface, it will Round droplets are formed due to surface tension. By the traction of the liquid by the dielectric force, the electrode will cause the contact angle of the two sides of the droplet to be asymmetric, which will cause the liquid to flow. Using electrode on-off switching, as shown in Figure 6 (a ~ e), the droplets can be separated, fused, and moved. They can be used for liquid transport and virus traction in the wafer. See Figure 7. COS ㊀ (V) -cose e〇 (V2) / 2 (rLG) t

Where 'r LG is the surface tension, the dielectric constant of the ε dielectric layer, ε0 is the dielectric dimension in vacuum,' t is the thickness of the dielectric layer, and 0 is the angle of liquid movement caused by the dielectric force. It can be known from the formula that if the efficiency of electrode traction is to be improved, the thickness of the dielectric layer must be reduced ft 'Select a dielectric layer with a large dielectric coefficient, or increase the applied electric C ° but the voltage applied to the right will make the overall device too high The cost increases, so it can be achieved by reducing the thickness of the dielectric layer and selecting materials with high dielectric constants. Guang Benming took advantage of this characteristic. After the separation mechanism separates the desired particles from each other, the traveling wave is used to separate them. Electricity

Page 25 200528179 V. Explanation of the invention (16) Wave-di electrophoretic force) The particles are drawn to the end of the separation electrode. The solution with the particles is formed into a droplet package by the above method, and a virus will be present in the droplet package. Then, the stylized electrode control can be used to precisely control the direction and speed of liquid movement, so that the liquid droplets can move in the hydrophobic area, so that the particles (including viruses) in the liquid can be pulled to the desired position. Figure 8 shows the electrode application method. As shown in Figure 8 (a), when the separation electrode separates the particles and uses the traveling wave dielectric power ^ "" 丨 丨 called — ^^ 一 (^ electrophoretic f0rce) to pull the particles to the separation electrode. Front end, where 0 and 1 represent the electrode current, 0 represents L0w, and 1 represents Higji. When the particles are concentrated at the forefront of the separation electrode, applying a voltage to the piezoelectric material 2 will cause the volume of the piezoelectric material to change. A squeezing force is applied in conjunction with the electrowetting-on-1ec tric method on the dielectric to apply the entire electrode as shown in Fig. 8 (dagger), which can compress the sample and attract it to the droplet package. In the hydrophobic channel, when the specimen enters the hydrophobic area ^ ', it will also bring particles into the hydrophobic area, and use the electrode to attract the droplets and then release the voltage of the piezoelectric material, so that the volume of the piezoelectric material returns to the original: At this time, the specimen will be pulled back to the separation electrode area, and the liquid absorbed by the droplet package will form a droplet package as shown in Figure 8 (c). Mingli ί ΐ ΐi: Each station has a medium solution with different conductivity. The hair Γ γΠ 蝌 is used to achieve the purpose of controlling the solution conductivity, as shown in Figure 8: / When the droplet is drawn to the next station, it will It has the effect of isolation from the surrounding solution and will not destroy the conductivity of the medium solution.

200528179 V. Explanation of the invention (17) Counting mechanism 1953 AD Coulter proposed that when a non-conductive particle flows through a thin capillary in a conductive solution, it will increase the resistance in the capillary. The size is related to the size of the non-conducting particles. The present invention uses this principle. As shown in Figure IX, it is a schematic diagram of a multi-ion channel counting device used in the present invention. It mainly uses one side of a plurality of nano-channels to place chlorination. Vaporized salts, such as potassium (KC1) or sodium chloride, guide the virus through the nanochannel. Because the virus has a blocking effect on the channel, the frequency of potassium ions flowing through the channel, that is, ion flow, is different, so it can be calculated. Number of passed viruses. The present invention also uses multiple channels to perform virus counting simultaneously, increasing the counting speed. Although the relationship between the change in conductivity (c 0 n d u c t a n c e) caused by particles flowing through the capillary is shown in the following formula:

△ g = 2 Ilr ^ r ^ r σ / L △ g: Conductivity change rate r: Particle radius σ: Solution conductivity L: Channel length Therefore, the chip will be used to change the disease when the virus passes through the nanometer flux, and therefore. When the virus first isolates the desired path, it will know the type of virus that the virus passed through due to the change in the resistance value, which will change and make the measured electricity meter reach the goal of counting.

Page 27 200528179 V. Description of the invention (18) [Channel particles When particles are in the channel, the lead electrode and the lower electrode 5 5 can be shown. The difference is that the columnar electric field of the ordering square is as strong as the electrode arrangement under the high-density electrode sub-channel. The electromagnetic simulation is shown in Figure 12. [Implementing the method of separating the electrode step 1, the glue substrate 6 2 step 2, the sub-flow sub-flow for weight and separation A variety of rows of particle migration and high density and electric field software outside the flow meter) | Rows of sub-channels, in a row of sub-types, electrode type, profitability, electric field, special electric zone, design path, find the electrically-completed electrode], when the order of the channel in the channel is better, the dielectric is generated and applied. Area 5 8: The field distribution is generated and will affect all particles. The electric field will be arranged outside the electrodes. The five real electric fields of the main thin film layer 5 6 of the current change mechanism must also be ineffective to flow the particles as shown in Figure 11 '. Attached to a stable sounding electric field molecules are directed in the direction of the ion and the design is such as the order 'so the structure is divided' and the electrode examples are oriented and electrically different. Relocate to as shown. The near velocity distribution situation is different. The design of the channels is different. Figure 13 and the stability of the relative arrangement of the upper electrodes 5 4 through the sorting of the channels are not shown. In a columnar electric field, particles 5 and 9 will pass through ion channels. ’The electric field must focus on the off direction, different. Through the present invention, the simulation results are shown. Formula] The plastic injection technology is used to shoot the flow channel and storage tank 67. Refer to Figure 14 (a). The photolithography is used and the sputtering machine is used.

Page 28 200528179 V. Description of the invention (19) " (sputter) Sputtering a layer of chromium (Cr) on the upper half of the substrate and then a layer of gold (Au) as the electrode layer 63, and use Li ft ff The process technology leaves the required pattern 'for the purpose of providing an electric field and an upper electrode for the measurement signal'. The main role of the chromium metal layer is to increase the adhesion between gold and the plastic substrate, so it does not need to be too thick; 3. Referring to FIG. 14 (b), the photolithography etching technology is used to leave the desired photoresist 61 as a spacer, as shown in FIG. 14 (c).

Step 4. Finally, coating — a thin layer of teflon 60 is used as the dielectric layer. Step 5. Referring to FIG. 14 (d), select ITO glass 66 as the sealing channel and storage tank 67. Material, because the ITO glass surface has a layer of transparent indium tin oxide 65, which can be used as an electrode; Step VI, referring to Figures 14 (e) and (f), using mechanical processing techniques such as spray shirts (document 'AN 〇ve 1 L 〇w L 〇sswafer-Leve 1 Packaging of the RF-MEMS Devices 20 02 IEEE) or laser perforation to make the required entry holes and escape holes on the IT〇 glass;

Step 7. Refer to FIG. 14 (S) 'Using the Lift off process technology, a layer of chromium (Cr) and a layer of gold (au) are used as electrode layers 68 on the back of the glass on the back of the glass, which is used for the electrode of the ion channel. For layer bonding; Step 8. Refer to Figure 14 (S) and cover it with a layer of teflon as the dielectric layer on IT0 glass. Step 9: Refer to Figure 14 (h) 'Use UV glue to process the glass and The base materials are connected to form a sealed state between the flow path and the storage tank 67.

200528179

[Electronic circuit of ion channel] The current flowing by ions through the nano channel depends on the pore size of the channel, and its current is about nano to hundreds of nano amps. Conventional large-scale instruments or electronic circuits. Reduced and integrated on the chip, the present invention redesigns it into an integrated circuit, description two; ΘΒ

Referring to Fig. 15 (a), the nano ampere current measurement electronic circuit can be roughly divided into five independent sub-circuits, including 1 · current / voltage converter (I-V converter) 2 · DC electric field voltage 3 · AC electric field voltage 4 Three-bit high-speed analog-to-digital converter 5 · Microcontroller, etc. · Low-noise operational amplifier used in a current / voltage converter, refer to Figure 15 (b) 'through-current amplification and a transimpedance amplification of a two-stage series circuit' whose input bias current is about 6 η A ~ 6 0 η A, bandwidth is about 10 μ〇 z gain is about lOMega ~ IMega.

Due to the current / voltage converter 71 used, the purpose is to convert a current signal of several nA to several hundreds of nA into a voltage of several mv. In this transfer amplifier circuit that requires gain, if a common oop amplifier is used for external appliances The structure of the group will use a very large resistance, which will inevitably occupy a large area during the waferization process. Therefore, in order to avoid the use of this large resistance, the traditional 0P op amp in voltage mode will not be used in the circuit structure, and the circuit structure with low input impedance will be replaced. The first stage current amplifier 711 amplifies currents ranging from several nA to hundreds of nA by 100-1000 times, and the second stage 712 uses a transimpedance amplifier to put about 100 thousand times, so the two stages are combined. The amplification gain is about 1 MegaVo1t / A.

Page 30 200528179 V. Description of the invention (21) A high-speed analog-to-digital converter 72 with two or more bits is used to convert multiple voltage levels and a reference level corresponding to different viruses, for example, If there are a total of six voltage levels when unblocked, it needs to be converted into a three-bit 'basic architecture as a flash circuit (refer to D. A. johns and K. Martin, 1997, Analog Integrated Circuit Design, pp · 50 7-5 1 3), the resistance between the levels is adjusted according to different viruses and the voltage level when it is not blocked. Its conversion speed is 100MHz-5 0 0MHz, which can be read by multiplexing. Multiple channels. The DC electric field voltage is 78. The purpose is to apply a DC bias voltage to the electrode so that the virus passes through the channel provided on the electrode in order to reduce the influence of noise (see Hami ii et. Al., 1981, Pfluegers Arch. Eur. J. Physiol. 391: 85-100) 'Provide a way to reduce the speed of virus passing through the nanopore, so that each virus can be suspended in the nanopore, not affected by diffusion, have time to let ions pass, basically control the sampling time At 1 microsecond, its voltage level is about 60-1 50mV. 'This DC electric field voltage can be applied via the current-to-voltage converter 7 丨 shown in Fig. 7 to generate a local electric field voltage through the current signal terminal I. In the second stage amplifier, the amplified value of the ion current is taken out by the differential amplifier.

The functions of the microcontroller 73 include: adjusting the voltage level and frequency of the AC electric field voltage, the level of the DC electric field voltage, the on-time and the off-time length of the synchronization signal, the communication with the two-bit high-speed analog digital converter, and the gene sequence The conversion and storage of the code, or the comparison with the gene sequence of the known disease stored in the memory 76, and display on the display (LCD) 74, or the temporary gene sequence " code, which is transferred through the RS-232 communication protocol 77, To an external computer 75. micro-

Page 31 200528179

The controller can also choose the number of viruses expanded by external memory and the size of the virus or determine the initial sale of the product, and the built-in, the readable channel δ has recorded a decline in the current level of the virus. Material memory 76 can also be read with another number and per channel, which can be used to compare virus contamination. [Production of ion channel 8〇] Step I, refer to the figure + >, the past and the last one, select ^ ten, one hundred first choose P-Type (100), thick $ h for the next low pressure on the substrate Chemistry Α / 〇.〇ΛΤ, Ν deposits a layer of silicon silicate (Si3N4) film 50nm-100nm on both sides of the soil material, as shown in Figure 16 (a); Second step > The photoetching technique draws out the pattern t on the upper half of the substrate = dry type, and then sets the substrate at 85. (The lice rolled potassium (K0H) was etched while stirring to the remaining film, and the characteristics of the single crystal silicon substrate 81 were used to etch a 54.74 degree beveled groove, as shown in Figure 16 (b); Step two, Next, a layer of silicon nitride (Si3N4) film of 200 nm to 25 nm is deposited on the substrate in the manner of Lpcvi), as shown in FIG. 16 (c); The lower half plane of the material is engraved with a pattern by means of dry etching, as shown in Figure 16 (d); Step five, the photolithography process is followed, and the lower plane of the substrate is sputtered by a sputtering machine. A layer of chromium (Cr) 83, and then a layer of gold (au) 84, as the lower electrode, as shown in Figure 16 (e); Step six, in the upper layer, the required thick film photoresist 8 5 As a mask, as shown in Figure 16 (f); and using a sputter on the substrate

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The upper plane of the substrate is sputtered with a layer of chromium (Cr) 83, and then a layer of gold (Au) 84, as the upper electrode, as shown in Figure 16 (g); remove the thick film photoresist, as shown in Figure 16 ( h); The electrodes on the upper plane are used to provide the electric field and the DC voltage, and the lower plane is used to measure the AC power pic. Grade Ampere Ion Current Measurement Circuit 'The main role of the chrome metal layer 83 is to increase the adhesion of gold to the silicon substrate 81, so it does not need to be too thick;' Step 7: The photolithography process is used in the lower layer. The required thick film photoresist ^ 5 is used as an insulating layer, as shown in figure sixteen (i);

Step 8. The film 82 is etched with dry money to leave about 70 0A, as shown in the step. Step 9. Use an e-beam writter to form a nano-channel 80 in the small hole and finally in the lower half of the nitride. Nitride stone around the lower layer (Si3N4) XVI (j). The photoresist on the lower half plane is used to make a monolithic wafer with a dry type on the nano-silicon film 82. Figure 17 is an exploded view of the monolithic wafer. The monolithic wafer contains an electrode port ion channel layer 100. A separate electrode is included in the electrode layer. Among them, 91 is a plastic substrate containing a flow channel and a storage tank 67.

^ Extreme! 63. The photoresist-made flow channel is used on the electrode layer. 61 ITO glass 66 is placed on the top, and 1ife-〇f bonding electrode 68 is used on the back of the glass. Figure 18 (b) is on glass 66. The electrode 68 made above is shown in the schematic diagram of the ion channel layer 10 (): Γ: Γ, Figure 18 is the second process of J: view, where 68 and 96 are mainly made by flip-chip packaging. Ripped and

200528179 V. Description of the invention (24) The electrodes on the road are bonded, and then the bottom filler is used to fill the gap to increase the bonding strength. 1 0 0 is an ion channel layer, and an electronic circuit 70 capable of calculating and measuring the number of viruses on the ion channel is shown in Fig. 18 (a). _ When the electrode 84 of the ion channel layer and the electrode 68 of the glass layer are joined, a sealed space of 67 will be formed between the upper and lower electrodes, as shown in Fig. 20, and Fig. 20 is a sectional view extending along section 19 of Fig. 19 (c). The present invention uses this method to create a flow channel in the ion channel. When the virus is drawn to the ion channel layer, it will flow through the nano channel (na η 〇-ρ 〇re) through the flow channel. Nano-pore can calculate the number of viruses in solution.

Page 34 200528179 Brief description of the diagram Figure 1 Flow chart of overall wafer detection Figure 2 Schematic diagram of separation technology Figure 3 Schematic diagram of virus pulling Figure 4 Schematic diagram of droplet packing Figure 5 Schematic diagram of counting mechanism Schematic diagram of electronic circuit Fig. 8 Intentional formation of droplet package Fig. 9 Flow chart of ion channel production Fig. 10 Schematic diagram of sorting and relocating electrode Fig. 11 Schematic diagram of columnar electric field Fig. 12 Simulation of electric field distribution Schematic diagram Figure 14 Overall chip package construction diagram Figure 15 Current amplification signal circuit diagram Figure 16 Ion channel process schematic diagram Figure 17 Schematic diagram of the overall chip explosion Figure 18 Schematic diagram of the cross section of the electrode package and the electrode package Figure 20 Wafer complete sectional view

Page 35

Claims (1)

200528179 VI. Scope of patent application1. A multi-channel high-sensitivity air suspension virus counting and detection device, mainly including-a set of virus capture mechanism including at least a set of vacuum pumping elements, an AC charging module, a static electricity Precipitation module; L-the so-called AC charging module, which is used to generate negative ions by AC discharge, so that the virus is sufficiently charged; '-the electrostatic precipitation module is a method that uses electrostatic precipitation to make high electrostatic fields Adsorb charged virus into pure water; A multi-layer wafer with one or more sets of independent special flow channels, each of which contains a set of separation mechanism and a nano-scale opening as an ion channel; the so-called separation mechanism includes at least A set of separation electrodes made by micro-electromechanical technology and at least one set of electrodes for generating droplet packets; a so-called separation electrode is used to apply alternating currents of different frequencies and amplitudes to make a specific virus due to the dielectric power ( dielectrophoresis f 〇I * ee) are different and separated; the so-called electrode for droplet encapsulation is the use of electric field switching to make the solution form droplets, One of the so-called nano-scale openings is to use micro-machining technology to make a thin film, and to open multiple nano-scale perforations on the thin film as ion channels, so that the thin film is interposed between rain micro-chambers. Ion solution in the micro-storage tank;-A set of n A (10-9 ampere) class ion current measurement circuit 'for a nanometer-level opening identified by the mother'-an independently set DC bias of the ion channel AC field of ion channel; Page 36 200528179 6. Scope of Patent Application-A Set of Controls § 'Used to control the magnitude and square of the DC bias of each so-called ion channel 1' Voltage and frequency of the AC electric field of the ion channel, and the amount of ion current The measured value is signal processed, and the number of viruses is calculated according to the number of current changes. 2 · The virus counting device according to item 1 of the stated patent scope, wherein the so-called ionization electrode is a staggered array electrode with different pitches, which mainly has different dielectric characteristics and conductivity between different viruses. The uneven electric field distribution generated by the array electrodes generates the attraction or repulsion of the dielectric force (DEP force), which separates different viruses on the separation device. 3. According to the virus counting device described in item 丨 of the scope of the patent application, straight = = = 'is mainly a method of appropriately configuring the electrodes on both sides of the flow channel =, and the surface is changed to make the surface of the flow channel hydrophobic, and report the solution When placed between two electrodes, an electric field is used; 'C does not transport droplets containing viruses in the flow channel. Tube knife-changing electrode array traction 4. As described in item 1 of the scope of the patent application, the virus counting penetrating, Gan & ion channel DC bias is based on the magnitude of the bias: square two: the so-called ice force, to Traction regulates the charged virus through the so-called: m :: generated electricity 5. The virus-counted ion channel AC electric field as described in the first item of the patent application scope is based on its voltage magnitude and frequency 200528179 VI. Scope of patent application _ Electrophoretic force to pull the bow 丨 Regulation of polarized virus through the so-called nanopore channel 6 · For example, the ionic current measurement circuit of the patent application scope No. 1 and 2 can be used to describe the virus count Device, which uses the required product of the so-called nano-scale openings, or the independent electromechanical manufacturing technology of a separate circuit is integrated in the same crystal. 7. The ion channel as claimed in the scope of the patent application] It is when the virus is regulating: several devices, the so-called next, one by one through the ion channel, g 2 ice /, /, or dielectric power to drive the plug, causing the ion current to drop. 17 meters channel, making the channel partially obstructed 8. As described in the scope of the patent application, the virus counts Meng Sanggan's virus, the type of which can be independently distributed in multiple groups according to the electrical properties of the virus. Tong Niu Sheng & added different separation electricity counts in the road. 〒 门 仃 仃 Same or different virus t-channel high-sensitivity air suspension virus counting * #The measurement method is mainly performed on a virus counting device, including the detection method, and the amount is measured by 5 / zt to SJ Ϊ: Dust and micro-particle loops in a fixed-time constant-flow part using vacuum pumping; bleed off the atmosphere; air-to-air pre-treatment (charging and electrostatic sinking) with a fixed volume for clothes and armors: viruses that circulate air, if necessary Page 38, 200528179 6. The scope of the patent application shall be exchanged first: make the charge wide enough, and then use the electrostatic sinking method to make the electrostatic field absorb the charged virus to pure water, and switch the electric field to change it! Move the traction virus-containing Liquid to the area of droplet = pole in order to automatically move the virus to the subsequent inspection and analysis device,-Virus separation: After entering the inspection and analysis device, the first stage will pass through the separation electrode, and a high frequency is applied to the electrodes staggered on both sides. The AC electric field causes a specific virus counted by a specific frequency to be "介" repelled to the center of the > channel, while others are not on the electrode, cut Change the contact of the electrode, so that electricity :: r :: will be adsorbed and set the virus along the flow path to encapsulate the droplet. 2 = ❹ Slow moving traction ~ Virus traction: Change the solution with the specific virus to make the solution form The liquid droplet is sealed, and then the liquid // is transferred to the electrode electrode area; Dagger sends the liquid droplet package to the ion channel ~ Secondary separation: After the first separation, if there is a little specific separation from the electrode area, in order to achieve The purpose of precise counting is to isolate the virus when the specific virus is dissipated via the traction electrode and the electrode when it is separated by the electric field, so it can be released if necessary. For artificial separation, firstly separate the electric field electrode of the separation electrode. Second: The electricity is separated from the separation and induction step. The remaining specific disease and disease are removed and the virus is attracted. The virus counts: when the disease number is in the ion channel; when f passes through the ion channel. Will block the channel part, page 39, 200528179 6. The scope of patent application causes the ion current to drop;-Analysis of test results: the number of times of current drop can be accurately calculated separately in multiple groups of independent tests Viral channel, in order to achieve the purpose of the virus count 0 Page 40