TR202021833A2 - DISPOSABLE PATHOGEN DETECTION CHIP AND A RELATED PRODUCTION METHOD - Google Patents

Abstract

The invention relates to a disposable pathogen detection chip (1) and a production method thereof. More specifically, the present invention provides the definitive existence of the microbiological structure by looking at the change in the emission optical power values, which is released as a result of the FRET (Förster Resonance Energy Transfer) event that occurs between two molecules, which allows fluorescent molecules with different wavelengths of energy to come together on the same target (pathogen). It relates to a disposable pathogen (any disease-causing organism: virus, bacteria, protozoa, prion, viroid or fungus) detection chip (1) and a method for producing it, which allows judgment on

Description

DESCRIPTION DISPOSABLE PATHOGEN DETECTION CHIP AND A RELATED PRODUCTION METHOD Technical Area The invention relates to a disposable pathogen detection chip and a manufacturing method thereof.

More specifically, the present invention relates to fluorescent molecules with different wavelengths of energy. FRET that occurs between two molecules, allowing them to come together at the target (pathogen) (Förster Resonance Energy Transfer) event resulting from the emission of optical power values It is the only one that allows to make a judgment about the definitive existence of the microbiological structure by looking at the change. use pathogen (any disease-causing organism: virus, bacteria, protozoa, prion, viroid or mushroom) detection chip and a method of producing it.

State of the Art Detection of the presence of the pathogen by performing the test in cases of microbiologically based epidemics The time elapsed between the two causes a delay in starting the treatment. Corona virus just As can be seen from today's conditions, where the struggle against various pathogens, including It may take about 1-2 days for the results to come out. For testing, from the patient's nose or Samples taken from the throat by swab method with special sticks are placed in special liquids (solutions). It is delivered to the authorized laboratory where the test will be performed. The samples were obtained by PCR method (Polymerase Chain). Reaction - Polymerase Chain Reaction; Even if the number of any virus or bacteria in the body is very low by duplicating the hereditary material of the pathogen, allowing it to be caught even an advanced diagnosis method) is examined and a final report is created. Although the samples Although it is a process of a few hours to examine and create the result report, the increased test due to the epidemic numbers delay the results considerably. This allows the presence of the pathogen to be detected quickly and sensitively. This highlights the need for a pathogen detection chip that will detect it somehow. in industrial and environmental samples replicating cells, especially microbial cells (e.g. from a solution to detecting, counting and identifying bacteria, fungi and molds is mentioned. However, here, the emission resulting from the FRET event for pathogen detection is optical. very briefly about the exact existence of the microbiological structure by looking at the change in power values. There is no solution that allows to reach a verdict in time. Detecting bat-borne coronavirus using real-time PCR under the title "time PCR" A method and kit for However, for pathogen detection, it is exposed as a result of the FRET event. The definite existence of the microbiological structure by looking at the change in the emission optical power values There is no solution that allows to reach a decision in a very short time.

As a result, it is necessary to detect the presence of the pathogen quickly and sensitively for early treatment. The need for the present invention has led to the emergence of the solution.

Purpose and Brief Description of the Invention The aim of the present invention is to target fluorescent molecules with different wavelengths of energy on the same target. FRET (Förster), which allows the (pathogen) to come together, thus occurring between two molecules Resonance Energy Transfer) is the result of the emission optical power values. It is the only one that allows to make a judgment about the definitive existence of the microbiological structure by looking at the change. The aim is to present a disposable pathogen detection chip and a method for its production.

It is a pathogen detection chip containing a chip body and fluorescent with different wavelengths of energy. to allow molecules to come together in the same target pathogen and thus create a bond between two molecules. As a result of the FRET event occurring, the emission resulting from the change in optical power values In order to allow a decision about the definitive existence of the microbiological structure, - at least one primary combined with a fluorescent dye that can be excitedly labeled at a certain wavelength - combined with a fluorescent dye that can be excitedly labeled at a different wavelength and at least one secondary probe used in conjunction with said primary probe to result in - at least one of said probes or pathogen coated on said chip body to the molecular structure that will immobilize it by covalently binding it on the chip body. a chemical that has contains.

In the preferred embodiment of the invention, the chemical mentioned is the silane molecule organic compound or THIOL molecule is its organic compound.

In a preferred embodiment of the invention, said chemical is composed of organic compounds of the silane molecule. One of them is THPMP (3-(Trihydroxysilyl)propyl methylphosphonate).

Said primary probes and secondary probes are antibodies/proteins selected for pathogen recognition. or nucleic acids (DNA/RNA) that have been designed and produced as a conjugate of pathogen nucleic acids oligomers or aptamers).

In case of applying the pass-through method in pathogen detection, the chip body is made of transparent material. It should be manufactured and preferably glass.

If the reflective method is used for pathogen detection, the chip body is chemically modified. It must be made of a resilient material.

In a preferred embodiment of the invention, tests are carried out in an easier and more practical way. to provide; with the pathogen detection chip in question mounted on the bottom of a cuvette is used.

In a preferred embodiment of the invention, said tub body is preferably cylindrical and It includes a cap with a hole at the top for the test stick to enter.

It is a pathogen detection chip production method in which fluorescent molecules with different wavelengths of energy are produced. to enable them to come together in the same target pathogen and thus By looking at the change in the emission optical power values released as a result of the FRET event, in order to allow the conclusion of the definitive existence of the microbiological structure; - the primary probe with fluorescent dyes that can be marked/marked by excitation at different wavelengths and preparation of dye-probe complexes by combining the secondary probe, - chemical coating of the chip body using the silanization method, - preferably with the primary probe combined with the fluorescent dye of the chip body coated with the chemical that the paint is coated on the chemically coated chip body, thus obtaining the pathogen detection chip, or The primary probe and secondary probe coupled with fluorescent dyes of the chip body coated with the chemical The paint is coated on the chemical coated chip body by connecting it with the probe, thus obtaining the pathogen detection chip In the preferred embodiment of the invention, to provide homogeneous distribution of the grab on the chip body. to; vacuum coating technique during the chemical coating process of the chip body is being implemented.

In order to preserve the three-dimensional structure of the probes; probes in the process of coating the chip body with paint and The chemical coated chip body is connected to each other by covalent bonds.

Preferred embodiment of the inventive pathogen detection chip production method MES preparation, PBS preparation, piranha preparation, alconox preparation, EDC/MES preparation, dye-probe complexes preparation and chip bodies, i.e. preferably cleaning the chip body with alconox, cleaning with acetone, ethanol cleaning, rinsing with distilled water, applying the first nitrogen drying process, piranha process, rinsing again with distilled water and the second nitrogen drying process. Surface cleaning and etching processes consisting of application processes, chemical coating of the chip body, whose surface cleaning and etching processes are completed, and then chip, preferably to increase the chemical and mechanical properties of the coating the coating and firing process, which consists of the firing of the body, measuring the contact angle of the chip body, which is coated and baked, Checking whether the angle value obtained is appropriate, if the angle value is appropriate continue operations, destroy the chip body if the angle value is not appropriate, and an optional control process consisting of starting a new one, In the control process, the chip body, whose angle value is found to be suitable, should preferably be combined with EDC/MES. surface functionalization process consisting of agitation, MES washing, Coating the chip body coming out of the surface functionalization process with paint and PBS adding, preferably washing with PBS to remove surface debris, preferably with a cuvette assembly and putting a certain amount of paint and PBS into the tubs. paint coating and tub assembly process, preferably multiple products of the end products that became the pathogen detection chip or kit in the previous step boxing and storage in which it is boxed and stored It is a pathogen detection method in which fluorescent molecules with different wavelengths of energy are the same target. to bring them together in the pathogen and thus the FRET event that occurs between the two molecules. As a result of the change in emission optical power values, the microbiological structure can be determined. in order to allow a judgment to be made about its definitive existence; - two probes with the primary probe attached to the chemical coated surface and the secondary probe free obtaining the name FRET as a result of interaction with the free pathogen, or - both the primary probe and the secondary probe attached to the chemically coated surface of the free pathogen obtaining the FRET name as a result of interaction with the probe, or - the pathogen is bound to the chemical coated surface and both free probes are connected to the pathogen obtaining the FRET name as a result of interaction or - with the free pathogen, preferably chemical, with the primary probe and the secondary probe free Obtaining the name FRET as a result of interaction on the coated surface is provided.

Measurement in both transmissive and reflective mode in the pathogen detection method of the invention can be done.

Brief Description of Figures Figure 1 is a workflow showing all the processes in the production process of the pathogen detection chip of the invention. sema is given.

A representative view of the pathogen detection chip mentioned in Figure 2a is given. In this view Since there is no pathogen in the environment, the FRET event does not occur.

Figure 2b gives another representative view of the pathogen detection chip in question. This Since it is pathogenic in the environment, the FRET event takes place. Because both probes are the same In this way, the two fluorescent substances in the two probes are combined, as they bind to the target pathogen. comes together. Excitedly labeled fluorescent substances at different wavelengths in the two probes They are called FRET due to the energy difference between them when they come side by side. The so-called energy transfer takes place.

Figure Ba shows a representative view of another embodiment of the pathogen detection chip. This Since there is no pathogen in the environment in appearance, the FRET event does not occur.

In the structuring mentioned in Figure Sb, the FRET event that occurs in case of pathogen in the environment. A representative view is given.

Figure 4a provides a representative view of another embodiment of the pathogen detection chip. This Since there is no pathogen in the environment in appearance, the FRET event does not occur.

In the configuration mentioned in Figure 4b, the FRET event that occurs in case of pathogen in the environment. A representative view is given.

A representative view of another alternative embodiment of the pathogen detection chip in Figure 5a. is given. In this view, since there is no pathogen in the environment, the FRET event does not occur.

In the configuration mentioned in Figure 5b, the FRET event that occurs in case of pathogen in the environment. A representative view is given.

In Figure Ba, the subject of the invention belongs to a preferred cuvette structure assembled with a pathogen detection chip. perspective view is given. The tub structure here is preferably round Iamels with a diameter of 15 mm. (pathogen detection chip) is designed accordingly.

Front section view of the said cuvette structure mounted with the pathogen detection chip in Figure Öb. is given.

In Figure 7a, another preferred cuvette structure assembled with the pathogen detection chip of the invention. perspective view is given. The tub structure here is preferably round with a diameter of 10 mm In Figure 7b, the front section view of the cuvette structure assembled with the pathogen detection chip is given.

Reference Numbers 1. Pathogen detection chip . chip body . Chemical . primary probe 40. Secondary probe 50. Bathtub 51. Body 52. Cover 53. Hole 60. Pathogen 70. Energy of excitation 80. FRET name 100. MES preparation 101. PBS preparation 102. Preparing Piranha 103. Alconox preparation 104. EDC/MES preparation 105. Preparation of dye-probe complexes 106. Inserting the lamellas into the holder 107. Cleaning with Alconox 108. Cleaning with acetone 109. Ethanol cleaning 110. Rinsing with distilled water 111. First nitrogen drying 112. Piranha process 113. Re-rinse with distilled water 114. Second nitrogen drying 115. Coating with chemicals 116. Baking 117. Contact angle measurement 118. Checking for suitability 119. Disposal and start over 120. Agitation with EDC/MES 121. Washing with MES 122. Coating paint and adding PBS 123. Washing with PBS 124. Assembling with the bathtub 125. Putting a certain amount of dye and PBS in the cuvettes 126. Boxing and storage in multiple products Detailed Description of the Invention The present invention shows that fluorescent molecules with different wavelengths of energy can be on the same target (pathogen (60)). FRET (Förster) that occurs between two molecules Resonance Energy Transfer) is the result of the emission optical power values. It is the only one that allows to make a judgment about the definitive existence of the microbiological structure by looking at the change. It relates to a disposable pathogen detection chip (1) and a method for producing it. subject of the invention Thanks to the pathogen detection chip (1), there is no need for diagnostic methods applied in laboratory environments. automatically and very briefly by measuring the change in emission optical power values without It is possible to detect the presence of pathogen (60) in a short time.

The pathogen detection chip (1), which is the subject of the invention, generally; chip body (10), said chip body (10) The chemical (20) coated on it is covalently attached to the chip body (10) coated with the said chemical (20). consists of primary probes (30) connected by a tie. Said pathogen detection chip (1) is preferably a It is used by being mounted inside the tub (50). In addition, the test kept in a special solution and The secondary probes (40) used for the test result are also the same test kit with the pathogen detection chip (1). is located in.

In the described configuration of the invention, the chip body 1D is transparent, preferably glass (lamella). material. In a different configuration of the invention, the chip body 10 is made of an opaque material. it is also possible. Chip at least one of said probes (30, 40) or pathogen (60) to coat the chip body (10). having a molecular structure that will immobilize it by attaching or embedding on its body (10) It is possible to use any chemical (20). Preferably silane (ing: silane - one side silicone any of its organic compounds or a THIOL compound can be used. In the preferred embodiment of the invention, this silane to coat the chip body 10 THPMP (3-(Trihydroxysilyl)propyl methylphosphonate) is one of its organic compounds. chemical (20) is used. THPMP is a liquid under normal conditions and one end is silane. It is a commercial product already available in the market, with a phosphonate molecule at the other end. However, this its use in this way has not been encountered before. Preferably as coating chemical (20) in the invention The reason for using THPMP; It can also bind any biological material to a surface. (bioconjugation feature) and other proteins other than those related to the target pathogen (60) It has resistance against sticking on the chip. In this way, the specific desired biological substance When connecting, it is possible to prevent unwanted materials from interacting with the chip. (THPMP is both a probe binder and a property that prevents foreign matter from attaching to the surface.) increases the selectivity of the chip). Also, THPMP can be done quickly in a few steps without an extra molecule. and allows high efficiency covalent chemical bonding. Recognizing the pathogen (60) binding of antibodies (antibody) with THPMP is found in the scientific literature, but pathogen (60) Its use as a FRET sensor in the detection of the invention was first revealed with the pathogen detection chip (1). is being placed.

In a preferred embodiment of the invention, the THPMP is vacuum deposition to the transparent chip body 10. (vacuum coating) method. For vacuum deposition, first of all, the liquid THPMP chemical (20) is taken into an isolated cabinet and a coarse vacuum is applied. Applied As the liquid vapor pressure drops as a result of vacuum, the THPMP liquid turns into gas, so the chip by condensing it again on its body (10), a regular coating is formed. of THPMP It is possible to coat the chip body (10) in liquid or in any other way. However, the most homogeneous distribution and the least negative effect on the transparency of the glass is the vacuum coating. It was concluded that it was realized by the deposition method. Vacuum in the vacuum deposition process amount, distance of glass from liquid, time etc. get the best result by optimizing the parameters possible. Pre-treating the chip surface with piranha solution (Sulfuric acidzHydrogen peroxide) cleaning also causes the silane molecules to attach to the glass by exposing the free hydroxyl groups in the glass. facilitates their attachment. Here, ultraviolet light and ozone cleaning as alternatives (UV/ozone) can also be applied. In addition, the vacuum deposition method can simultaneously remove hundreds of chip bodies. (10) presents a method for coating, a solution that has not been applied in the literature before.

In a preferred embodiment of the invention, the chip body 10 is combined with the organic compound of the silane molecule. After coating, the chip body is used to increase the chemical and mechanical resistance of the coating. (10) are kept in a vacuum oven at high temperature. So that the coating is attached to the chip body (10) Stable by strengthening the adhesion (with washing etc. unless we scrape it mechanically) It is aimed to obtain a non-corrosive coating. The quality of the coating is the contact angle (contact angle) can be measured and evaluated. Contact with the water droplet in the preferred configuration Angle of at least 55° is required to proceed to the next step.

As a next step, fluorescent dyes that can be excited at a certain wavelength before primary probes (30) (antibody/protein) coupled with covalently using chemical linkers It is attached to the top of this coating. These primary probes (30) fluoresce before excitation. (The probes to be used for the detection of pathogen (60) cannot be used for FRET. combined with suitable fluorescent dyes) to obtain a fluorescent coating on the chip body (10). is being done. Fluorescent coupled primary probes (30) were used using chemical methods. Since it is covalently bound to the surface coating molecules (Preferably THPMP) and interacts with the chip body. (10) the structure of the probes remains as a stable structure without deteriorating since it does not interact directly is provided. Antibody, protein etc. Any probe such as When it sticks, its three-dimensional structure can be disrupted because it interacts with the community, while the subject of the invention In our solution, the probes are made of glass (or The antibody/proteins are prevented from interacting with any other chip body material. dimensional structure is preserved.

Chemically coated (20) followed by fluorescent coupled primary probes (30) The chip bodies (10) that are stained with (antibody/protein) are ready for use. find it In the preferred embodiment, the chip body (10) that is ready for use is preferably cylindrical. It is provided with a tub (50) and the tests are carried out in an easier and more practical way.

For this, a ready-to-use chip is placed on the lower part (base) of the body (51) of the cuvette (50). cover (52) with a hole (53) on the top of which the test stick can be inserted while the body (10) is being attached. It can be positioned or manufactured integrally with the body. So the test will be done The swab taken from the person with the test stick is placed on the pathogen detection chip (1) in the cuvette (50). is being transported.

To be used with the pathogen detection chip (1) to detect the presence of the pathogen (60) Secondary probes (40) (antibody/protein) suspended in solution were previously stained with fluorescent dye. coupled and at a certain wavelength (but different from that excited by the primary probe 30) again, the primary probe (30) is a wavelength that can be excited by the wavelength of fluorescent light (emission) required) is marked with a warning.

The sample taken from the person to be tested is placed on the pathogen detection chip (1). After binding the pathogen (60) particle or target molecule to the primary probes (30), the solution The secondary probes (40) kept in the solution are placed on the pathogen detection chip (1). is pouring. Under normal conditions, the coexistence of these two probes is unlikely. But every Since both probes have affinity for the pathogen (60), this can be done in an environment where the pathogen (60) is present. two probes are next to each other (both probes connect to the same pathogen (60)) and thus two fluorescent substances are juxtaposed. Different wavelengths of fluorescent substances in the two probes Due to the fact that they are marked by warning, when they come side by side, they are aware of the energy difference between them. Therefore, the energy transfer called FRET takes place.

The pathogen detection chip (1) is powered by an excitation energy (70) according to the wavelength of the primary probe (30). is warned. For this reason, while the pathogen (60) is absent, the fluorescent name comes from the primary probe (30) in the environment. In case of pathogen (60), it can also be removed from the secondary probe due to the energy transfer (FRET) between them. In (40) the fluorescent name (FRET designation (80)) is displayed. Energy transfer is only possible in the environment. (60) in this way, the presence of the pathogen (60) both very sensitively. It can be measured and because this energy transfer does not occur when something other than the pathogen (60) is present. It is ensured that there are no false positive measurements.

To summarize briefly, first of all, the chip body (10) is coated with the organic compound of the silane molecule, pathogen detection chip (1) by attaching this chemical coating (20) with primary probes (30). and finally pathogen (60) containing/free swab together with secondary probes (40) If there is a pathogen (60) in the environment, via the energy transfer (RFET) of this chip, by emitting fluorescent light, namely FRET (80) at a wavelength that it would not normally emit. the presence of the pathogen (60) is detected.

In our invention, coupling of fluorescent dyes and probes was performed using standard techniques. is provided. Fluorescent dyes are excited by laser source. Connected to primary probe (30) The fluorescent dye attached to the secondary probe (40) with the fluorescent dye is at different wavelengths (ie, different energy levels) are stimulated. As the wavelength increases, the excitation energy decreases. fluorescent dyes When excited at one wavelength, they radiate at a lower energy wavelength.

Fluorescent dyes are much smaller and simpler molecules than probes (antibody).

Since fluorescents have a chemical affinity for the probes, they can be attached to the probes. Painted and the excitation labeled primary probes (30) are attached to the chip body coated with the organic compound of the silane molecule. (10) is connected with a covalent bond to preserve the three-dimensional structure of the probes. your probes Normally they do not have the ability to make radiation, this feature is attached to the probes by binding with fluorescent molecules. is gained. Antibodies (probes) are pathogen (60) specific, they recognize pathogens (60) and it can be connected to their specific area. Briefly, probes when pathogen (60) is present in the environment binds to the pathogen (60).

Antibody selection for pathogen (60) recognition in probe selection for the pathogen detection chip (1) of the invention has been made. Considerations when choosing antibodies (in particular for COVID-19): - Confirmed by the World Health Organization that it can be used in the diagnosis of COVID-19, - Verified by the manufacturer that it can be used as a diagnosis, - Differential systems that can recognize different regions on the outer surface of the virus that causes COVID-19 disease have antibodies, - The amount of purchased antibody can be diluted in small proportions.

In the selection of fluorescent dye, the criterion of high efficiency binding of the dye to the probe (antibody/protein) is prerequisite. is in the background. Starting to work with appropriate antibody labeling kits in FRET application and After the successful result is obtained, a lower cost is required to bind the probe and the fluorescent dye. It is planned to investigate cost-effective methods. In this direction, initially ultraviolet/blue a FRET pair (two dyes) that can be excited in colors and whose FRET name wavelength is blue/green use is preferred.

Although the system is optimized for antibody/protein probes, pathogen (60) Nucleic acids (DNA/RNA) designed and produced as complementary nucleic acids oligomers or aptamers) can likewise be used as probes by binding to FRET pairs.

In this case, the primary nucleic acid bound to the first FRET dye will be covalently bound to the surface, a secondary nucleic acid bound to the second FRET dye will be used with the sample. If pathogenic nucleic acids are detectable by nucleic acid probes, the FRET dye pair is will come together and the name FRET (80) will take place.

The production steps of the inventive pathogen detection chip (1) are given below in more detail.

Most of the steps are chemical (20) coating and staining (primary probes 30) over coating. It is aimed at increasing the quality of the bonding process. For this reason, coating and painting Such steps to improve the quality of the inventive pathogen detection chip (1) They are not indispensable steps for production and it is possible to apply them with different methods.

Thanks to these steps to increase the quality, the pathogen detection chip (1), which is the subject of the invention, has a pathogen (60) It is provided to increase the sensitivity of detection.

The production method of the pathogen detection chip (1) consists of 7 steps. These; - Preliminary stage - Surface cleaning and etching processes - Coating and firing process - Control process - Surface functionalization process - Paint coating and tub assembly process - Boxing and storage process Preliminary actions (without order); MES preparation (100), PBS preparation preparation of probe complexes (105) and insertion of chip bodies (10), ie Iamels, into the holder (106) is in the form.

Surface cleaning and etching processes are respectively; cleaning the chip body (10) with alconox (107), cleaning with acetone (108), cleaning with ethanol (109), rinsing with distilled water (110), first applying the nitrogen drying (111) process, putting it into the piranha process (112), reconstituting with distilled water. rinsing (113) and applying the second nitrogen drying (114) process. Surface cleaning and The chip to be used in the measurement using etching processes and chemical or physical methods bodies (10) (eg, glassware) are cleaned. The aim here is to make chemical molecules the fine particles formed by the dusts that are around under normal conditions, which will enable it to bind. is to reveal the hydroxyl groups hidden under a carbon layer.

Coating and firing process respectively; The chip whose surface cleaning and etching processes have been completed coating (115) of the body (10) with a chemical (preferably organic compound of the silane molecule) followed by firing (116). Both bioconjugation and non-specific interaction to coat THPMP chemical (20) is preferred because of its resistance feature and it is converted to gas phase in vacuum oven. organic coating is done. Using the silanization method for the coating process (Chemical (20) compounds with reactive groups on them, a method called silanization is attached to the glass surfaces by means of vacuum coating technique is applied. This transaction carried out under vacuum. After chemical coating (115) of the chip body (10) The chemical and mechanical properties of the coating by applying heat treatment with firing (116) It is aimed to increase it, thus making it more durable. The baking (116) process is preferably carried out in the form of vacuum firing.

In order of control process; The contact angle of the chip body (10) in which the coating and firing process takes place Making the (contact angle) measurement (117), checking whether the obtained angle value is appropriate or not. (118), continue operations if the angle value is appropriate, if the angle value is not appropriate, the chip body (10) and starting a new one (119). Preferably 55° and above angle values are considered appropriate, values below are considered inappropriate.

Surface functionalization process, respectively; The chip whose angle value is found appropriate during the control process rinsing the body (10) with EDC/MES ( (preferably dripped MES) with) form. EDC is intermediate in antibody binding to THPMP during the next paint coating process. It acts as a molecule (mediator). MES, on the other hand, shows that this reaction takes place in high yield, It is a stable pH buffer solution.

Paint coating and tub assembly process, respectively; chip from surface functionalization process coating the body (10) with paint and adding PBS (122) with PBS for cleaning surface debris. washing (PBS, the 3D structures of the antibodies are intact) It is a salt and buffer solution with a pH value of 7.4 and similar to physiological conditions). assembly (124) (preferably sealed system), a certain amount of paint and PBS in the tubs The insertion is (125). Before all this paint coating and tub assembly process for pathogen (60) detection in the preparation (105) process of dye-probe complexes in preparation The probes to be used are combined with fluorescent dyes suitable for FRET. Fluorescent The primary probes (30), whose coupling and excitation take place, are chipped using chemical methods. covalently to the intermediate molecules on the coating (preferably THPMP) on the body (10) With the connection of the above-mentioned paint coating process is realized. Thus the only A disposable pathogen detection chip (1) is obtained. To make it more useful (e.g. kit) It is assembled with a bathtub (50).

In the boxing and storage process; which becomes the pathogen detection chip (1) or kit in the previous step The end products are preferably boxed and stored as multiple products (126).

Although the structure, production steps and detection of the pathogen of the pathogen detection chip (1), which is the subject of the invention, Although it is within the framework of the details described above, there are different alternative possibilities for them. (configurations) are also available.

In the preferred embodiment described in the specification, the primary probe (30) is attached to the chemical (20) coated surface. and both when the secondary probe (40) is free (not attached to the chemical (20) coated surface) As a result of its interaction with the free target pathogen (60), the name FRET (80) is formed. the presence of the pathogen (60) is detected. A representative view of this is given in Figure 2b.

In an alternative embodiment of the invention, the primary probe 30 and the secondary probe 40 are applied to the chemically coated surface. As a result of the interaction of the free target pathogen (60) with both probes, FRET The presence of the pathogen (60) is detected by the occurrence of the name (80). A representation of this view is given in Figure 3b.

In another alternative embodiment of the invention, the target pathogen (60) binds to the chemical (20) coated surface. free primary probe (30) and secondary probe (40) (both probes) with pathogen (60) As a result of the interaction, the presence of the pathogen (60) is detected by the formation of the name FRET (80). is being done. A representative view of this is given in Figure 4b.

In another alternative embodiment of the invention, the primary probe 30 and the secondary probe 40 are free. surface (preferably chemically (20) coated surface) with both free target pathogen (60) The presence of the pathogen (60) is detected by the formation of the name FRET (80) as a result of interaction with is being done. A representative view of this is given in Figure 5b.

With the pathogen detection chip (1) that is the subject of the invention, both transmission and reflection measurement can be made. In the case of measuring in the plug-in mode, the chip body (10) Measurement in reflective mode when it should be of transparent material and preferably glass In the case where the chip body (10) is of a material that can be chemically modified, required.

Finally, the molecule that will bind at least one of the probes or pathogen (60) onto the chip body (10). chemical (20), pathogen (60), which has a structure and is used to cover the chip body (10) Although it is not an essential feature for the detection of pathogen (60), it increases the efficiency in the detection of pathogen (60). Therefore, its use is very important.

Claims (1)

REQUESTS It is a pathogen detection chip (1) containing a chip body (1D), and its feature is; In order to allow fluorescent molecules with different wavelengths of energy to come together in the same target pathogen (60), and thus to allow a judgment on the precise existence of the microbiological structure by looking at the change in the emission optical power values released as a result of the FRET event occurring between the two molecules; - at least one primary probe (30) combined with an excitation-labelable fluorescent dye of a certain wavelength, - at least one secondary probe (40) combined with an excitable-labeled fluorescent dye of a different wavelength and used in conjunction with said primary probe (30) to conclude the test ), - containing a chemical (20) coated on the said chip body (1D) and having a molecular structure that will immobilize at least one of the said probes (30, 40) or the pathogen (60) by attaching or embedding it on the chip body (10). is to be characterized. It is a pathogen detection chip (1) according to claim 1, and its feature is; characterized by the said chemical (20) being a silane molecule organic compound or THIOL molecule organic compound. It is a pathogen detection chip (1) according to claim 2, and its feature is; characterized by the chemical (20) being THPMP (3-(Trihydroxysilyl)propyl methylphosphonate), one of the organic compounds of the silane molecule. A pathogen detection chip (1) according to any of the previous claims, and its feature is; said primary probes (30) and secondary probes (40) are antibodies/proteins selected for recognizing the pathogen (60) or nucleic acids (DNA/RNA oligomers or aptamers) designed and produced as a conjugate of pathogen (60) nucleic acids. A pathogen detection chip (1) according to any of the previous claims, and its feature is; in the case of applying the pass-through method in pathogen detection, the chip body (10) is made of transparent material and preferably glass. It is a pathogen detection chip (1) according to any of the claims 1-4, and its feature is; if the reflective method is used in pathogen detection, the chip body (10) is made of a material that can be chemically modified. A pathogen detection chip (1) according to any of the previous claims, and its feature is; in order to make the tests easier and more practical; it is characterized in that said pathogen detection chip (1) is used mounted on the bottom of a cuvette (50). It is a pathogen detection chip (1) according to claim 7, and its feature is; It is characterized in that the body (51) of said cuvette (50) is preferably in cylindrical form and includes a cover (52) having a hole (53) on the upper side where the test rod can enter. It is a pathogen detection chip (1) production method, and its feature is; In order to allow fluorescent molecules with different wavelengths of energy to come together in the same target pathogen (60), and thus to allow a judgment on the precise existence of the microbiological structure by looking at the change in the emission optical power values released as a result of the FRET event occurring between the two molecules; Preparation of dye-probe complexes by combining the primary probe (30) and the secondary probe (40) with fluorescent dyes that can be excited/marked at different wavelengths (105), coating the chip body (10) with chemical (20) using the silanization method (115), preferably By connecting the chip body (10) coated with the chemical (20) with the primary probe (30) combined with the fluorescent dye, the dye is coated on the chip body (10) coated with the chemical (20), thus obtaining the pathogen detection chip (1) or with the chemical ( 20) by connecting the coated chip body (10) with the primary probe (30) and secondary probe (40) combined with the fluorescent dyes, the dye is coated on the chip body (10) coated with the chemical (20), thus obtaining the pathogen detection chip (1) It is characterized by the fact that it includes process steps. It is a pathogen detection chip (1) production method according to claim 9, and its feature is; in order to ensure the homogeneous distribution of the grab on the chip body (10); It is characterized by the application of vacuum coating technique during the chemical coating (115) process of the chip body (10). It is a pathogen detection chip (1) production method according to claim 9 or 10, and its feature is; to preserve the three-dimensional structure of the probes; It is characterized by the covalent bonding of probes and chemically coated chip body (10) to each other in the process of coating the chip body (10) with paint. 12. A pathogen detection chip (1) production method according to any one of claims 9-11, and its feature is; Preliminary preparation stage consisting of MES preparation (, preparation of alconox (, preparation of dye-probe complexes (105) and placing of chip bodies (10) i.e. gels in the holder (106)), preferably cleaning the chip body (10) with alconox (107), with acetone cleaning (108), cleaning with ethanol (109), rinsing with distilled water (110), applying a first nitrogen drying (111), piranha or UV/ozone treatment (112), re-rinsing with distilled water (113), and The surface cleaning and etching processes consisting of the application of the second nitrogen drying (114) process, the surface cleaning and etching processes are completed, the chip body (10) is coated with chemicals (115), and then the chip body (10) is baked (preferably to increase the chemical and mechanical properties of the coating). 116), the coating and firing process consisting of processes, measuring the contact angle (117) of the chip body (10) where the coating and firing process is carried out, checking whether the angle value obtained is appropriate (118), continuing the processes if the angle value is appropriate, if the angle value is If it is not suitable, an optional control process consisting of destroying the chip body (10) and starting a new one (119), shaking the chip body (10) whose angle value is found suitable during the control process, preferably with EDC/MES ( the surface functionalization process consisting of operations, from the surface functionalization process Paint coating and cuvette mounting process, consisting of coating the resulting chip body (10) with paint and adding PBS (preferably mounting with the cuvette (124) and adding a certain amount of paint and PBS to the cuvettes (125), - pathogen detection chip in the previous step (1 ) or the final products, which become kits, are preferably boxed and stored as multiple products (126) and include boxing and storage process steps. It is a pathogen (60) detection method and its feature is; In order to allow fluorescent molecules with different wavelengths of energy to come together in the same target pathogen (60), and thus to allow a judgment on the precise existence of the microbiological structure by looking at the change in the emission optical power values released as a result of the FRET event occurring between the two molecules; Two probes interact with the free pathogen (60) with the primary probe (30) attached to the chemical (20) coated surface and the secondary probe (40) free, resulting in the FRET name (80) - primary probe (30) and secondary probe (40) obtaining the name FRET (80) when the free pathogen (60) interacts with both probes while attached to the chemical (20) coated surface, or - FRET as a result of the pathogen (60) binding to the chemical (20) coated surface and interacting with the pathogen (60) of both free probes It is characterized by obtaining the name (80) or - obtaining the FRET name (80) as a result of the interaction of the primary probe (30) and the secondary probe (40) with the free pathogen (60) when they are in their free state, preferably on the surface coated with the chemical (20). It is a pathogen (60) detection method according to claim 13, and its feature is; It is characterized by the fact that it can be measured in both transmissive and reflective mode.