WO2022139706A1 - A single-use pathogen detection chip and a production method thereof - Google Patents
A single-use pathogen detection chip and a production method thereof Download PDFInfo
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- WO2022139706A1 WO2022139706A1 PCT/TR2020/051423 TR2020051423W WO2022139706A1 WO 2022139706 A1 WO2022139706 A1 WO 2022139706A1 TR 2020051423 W TR2020051423 W TR 2020051423W WO 2022139706 A1 WO2022139706 A1 WO 2022139706A1
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- pathogen
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Definitions
- the present innovative pathogen detection chip (1) generally includes; a chip body (10), a chemical (20) that is coated on mentioned chip body (10), primary probes (30) that are bonded (preferably by covalent bond) to said chip body (10) coated by mentioned chemical (20). Mentioned pathogen detection chip (1) is preferably used by being assembled in a cuvette (50). In addition, secondary probes (40) which are kept in a special solution and used for finalization of the test are included in the same test kit together with said pathogen detection chip (1).
Abstract
The invention relates to a single-use pathogen detection chip (1) and a production method thereof. More specifically, the present invention relates to a single-use pathogen (any organism having capable to cause a disease: virus, bacteria, protozoa, prion, viroid, or fungus) detection chip (1) which enables fluorescent molecules having energy in different wavelengths from each other to interact on the same target (pathogen (60)) thus, allows to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET (Forster Resonance Energy Transfer) event occurred between two molecules, and a production method thereof.
Description
A SINGLE-USE PATHOGEN DETECTION CHIP AND A PRODUCTION METHOD THEREOF
Technical Field
The invention relates to a single-use pathogen detection chip and a production method thereof.
More specifically, the present invention relates to a single-use pathogen (any organism having capable to cause a disease: virus, bacteria, protozoa, prion, viroid, or fungus) detection chip which enables fluorescent molecules having energy in different wavelengths from each other to interact on the same target (pathogen) thus, allows to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET (Forster Resonance Energy Transfer) event occurred between two molecules, and a production method thereof.
Prior Art
In microbiological-based pandemics or epidemics, the time passed between testing and detecting the presence of pathogen causes a delay in starting treatment. As seen from today’s conditions where people struggle against various pathogens, especially the coronavirus, it can take about 1-2 days for test results to be obtained. Fluid samples taken for test from the patient’s nose or throat with special swabs by using the swab method are delivered in special liquids (solutions) to the authorized laboratory where the test will be performed. The samples are analyzed by PCR method (Polymerase Chain Reaction; An advanced diagnostic method that allows any virus or bacteria to be caught even the number of them is very few in the body by duplicating the hereditary material of pathogen) and a final test report is created. Although the examination of the samples and preparation of the final test report takes a few hours, the number of tests increased due to the pandemic causes the results to delay considerably. This situation emerges out the require for a pathogen detection chip that will quickly and precisely detect the presence of the pathogen.
The patent document no US9290382B2 mentions a solution for detection, enumeration, and identification of cells (e.g., bacteria, yeasts, and molds) in medical, industrial, and environmental samples, under the title “Rapid detection of replicating cells”. However, here, for the pathogen detection, there is not any solution that allows a judgment on the exact existence of the microbiological structure in a very short time by observing the change in the emission optical power values resulting from the FRET event.
In the patent document KR102018201 B1 , a method and kit for detecting bat-derived coronaviruses by using real-time PCR is described under the title “Method and kit for detecting batcoronavirus using real-time PCR”. However, here, for the pathogen detection, there is not any solution that allows a judgment on the exact existence of the microbiological structure in a very short time by observing the change in the emission optical power values resulting from the FRET event.
As a result, the requirement for a rapid and precise detection of the presence of the pathogen for early treatment has led to the present innovative solution to emerge.
Objectives and Short Description of the Invention
The aim of the invention is to present a single-use pathogen detection chip that enables fluorescent molecules having energy in different wavelengths from each other to interact (come together) on the same target (pathogen) thus, allows to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET (Forster Resonance Energy Transfer) event occurred between two molecules, and a production method thereof.
A pathogen detection chip which, comprising a chip body and, in order to enable fluorescent molecules having energy in different wavelengths from each other to interact on the same target pathogen and thus, to allow to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET event occurred between two molecules; it comprises
- at least one primary probe bonded with a fluorescent dye that can be marked by being excited at a certain wavelength,
- at least one secondary probe which is bonded with a fluorescent dye that can be marked by being excited at a different wavelength and, which is used together with mentioned primary probe for resulting the test,
- a chemical that is coated on the chip body and has the molecular structure that immobilizes at least one of said probes or pathogen by bonding or embedding them on the chip body.
In the preferred embodiment of the invention, mentioned chemical is the silane molecule organic compound or THIOL molecule.
In the a preferred embodiment of the invention, said chemical is THPMP (3-(Trihydroxysilyl)propyl methylphosphonate) which is one of the silane molecule organic compounds.
Mentioned primary probes and secondary probes are antibodies/proteins selected to recognize the pathogen or, nucleic acids (DNA/RNA oligomers or aptamers) that are designed and produced as complementary of pathogen nucleic acids.
In the case of using optical transmission mode in pathogen detection, the chip body should be made of transparent material, preferably made of glass.
In the case of using optical reflection mode in pathogen detection, the chip body should be made of a chemically modifiable material.
In the a preferred embodiment of the invention, the pathogen detection chip is used as being assembled on the bottom of a cuvette in order to allow the tests to be made easier and more practically.
In the a preferred embodiment of the invention, the body of mentioned cuvette is preferably in cylindrical form and on its upper side, it comprises cover including a hole in which swab can enter.
A pathogen detection chip production method and, in order to enable fluorescent molecules having energy in different wavelengths from each other to interact on the same target pathogen and thus, to allow to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET event occurred between two molecules; it comprises the following steps: preparation of dye-probe complexes by bonding the primary probe and secondary probe with fluorescent dyes that were marked/can be marked by being excited at different wavelengths, coating of chip body with chemical by using silanization method, preferably, coating dye onto the chemical-coated chip body by bonding of the fluorescent- dye-bonded primary probe onto the chemical-coated chip body, thus obtaining pathogen detection chip or, coating dye onto the chemical-coated chip body by bonding of the fluorescent-dye-bonded primary and secondary probes onto the chemical-coated chip body, thus obtaining pathogen detection chip.
In preferred embodiment of the invention, vacuum coating technique is applied during the coating process of the chip body with chemical in order to ensure homogeneous distribution of the coating on the chip body.
In order to preserve the three-dimensional structure of probes; probes and the chemical coated chip body are bonded to each other by covalent bond in the dye coating process of the chip body.
A preferred embodiment of the present innovative pathogen detection chip production method comprises the following steps: preliminary preparation phase consisting of MES preparation, PBS preparation, piranha preparation, alconox preparation, EDC/MES preparation, preparation of dye-probe complexes and, positioning chip bodies (namely slides) within holder, surface cleaning and abrasion processes consisting of the following steps: preferably cleaning of the chip body with alconox, cleaning the chip body with acetone, cleaning the chip body with ethanol, rinsing the chip body with distilled water, applying first drying operation with nitrogen, continuing with piranha or UV/ozone process, re-rinsing the chip body with distilled water, applying second drying operation with nitrogen, coating and oven curing processes consisting of the following steps: coating the chip body whose surface cleaning and abrasion processes are completed, with chemical (preferably silane molecule organic compound) and then, preferably oven curing the coated chip body in order to increase the chemical and mechanical properties of the coating, an optional control process consisting of the following steps: measuring contact angle of the chip body whose coating and oven curing processes are completed, controlling whether the obtained contact angle is proper or not, continuing with next step if the contact angle value is proper, if the contact angle is not proper extirpating the chip body and starting with new one, surface functionalization process consisting of the following steps: preferably agitation of the chip body whose contact angle was determined as appropriate in the control process with EDC/MES on a shaker, washing with MES, dye coating and cuvette assembly process consisting of the following steps: coating of the chip body exiting from the surface functionalization process with dye and adding PBS, preferably washing with PBS in order to remove surface waste, preferably assembling with cuvette, putting a certain amount of dye and PBS within cuvettes,
packaging and storing process in which the final products that become pathogen detection chip or kit in the previous step are preferably packaged as multiple products and stored.
A pathogen detection method and, in order to enable fluorescent molecules having energy in different wavelengths from each other to interact on the same target pathogen and thus, to allow to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET event occurred between two molecules; in the case of that the primary probe is bonded to the chemical coated surface and the secondary probe is free, obtaining FRET emission resulting from the interaction of two probes with the free pathogen or, in the case of that the primary probe and the secondary probe are bonded to the chemical coated surface, obtaining FRET emission resulting from the interaction of two probes with the free pathogen or, obtaining FRET emission resulting from the interaction of two free probes with the pathogen that is bonded to the chemical coated surface or, obtaining FRET emission resulting from the interaction of two free probes with the free pathogen preferably on the chemical coated surface.
The present innovative pathogen detection method allows to make measurement in both transmission mode and reflection mode.
Description of the Figures
In Figure 1 , a process flow chart showing all processes in the production of the present innovative pathogen detection chip is given.
In Figure 2a, a representative view of mentioned pathogen detection chip is given. In this representative view, the FRET event does not occur since there is no pathogen in the environment.
In Figure 2b, another representative view of said pathogen detection chip is given. In this representative view, the FRET event occurs since there are pathogens in the environment. Since the two probes come together by bonding on the same target pathogen, the two fluorescent substances in the two probes come together too in this way. Since the fluorescent substances in the two probes are marked by being excited at different wavelengths, when they come together, the energy transfer called FRET occurs due to the energy difference between them.
In Figure 3a, a representative view of another embodiment of the pathogen detection chip is given. In this representative view, the FRET event does not occur since there is no pathogen in the environment.
In Figure 3b, a representative view of the FRET event that occurs in the mentioned embodiment (given in 3a) in the case if there is pathogen in the environment is given.
In Figure 4a, a representative view of other alternative embodiment of the pathogen detection chip is given. In this representative view, the FRET event does not occur since there is no pathogen in the environment.
In Figure 4b, a representative view of the FRET event that occurs in the mentioned embodiment (given in 4a) in the case if there is pathogen in the environment is given.
In Figure 5a, a representative view of another alternative embodiment of the pathogen detection chip is given. In this representative view, the FRET event does not occur since there is no pathogen in the environment.
In Figure 5b, a representative view of the FRET event that occurs in the mentioned embodiment (given in 5a) in the case if there is pathogen in the environment is given.
In Figure 6a, a preferred cuvette structure assembled with the present innovative pathogen detection chip is given. Mentioned cuvette structure is preferably designed suitable for round slides (pathogen detection chip body) with a diameter of 15 mm.
In Figure 6b, a front section view of said cuvette structure (given in 6a) assembled with the present innovative pathogen detection chip is given.
In Figure 7a, a perspective view of another preferred cuvette structure assembled with the present innovative pathogen detection chip is given. Mentioned cuvette structure is preferably designed suitable for round slides (pathogen detection chip body) with a diameter of 10 mm.
In Figure 7b, a front section view of said cuvette structure (given in 7a) assembled with the present innovative pathogen detection chip is given.
Reference Numbers
1. Pathogen detection chip
10. Chip body
20. Chemical
30. Primary probe
40. Secondary probe
50. Cuvette
51. Body
52. Cover
53. Hole
60. Pathogen
70. Excitation energy
80. FRET emission
100. MES preparation
101. PBS preparation
102. Piranha preparation
103. Alconox preparation
104. EDC/MES preparation
105. Preparation of dye-probe complexes
106. Positioning glass slides (chip bodies) within holder
107. Cleaning with Alconox
108. Cleaning with Acetone
109. Cleaning with Ethanol
110. Rinsing with distilled water
111. First drying with nitrogen
112. Piranha process
113. Re-rinsing with distilled water
114. Second drying with nitrogen
115. Coating with chemical
116. Oven curing
117. Measuring contact angle
118. Controlling whether the angle is proper or not
119. Extirpating and starting with new one
120. Agitation with EDC/MES on a shaker
121. Washing with MES
122. Coating with dye and adding PBS
123. Washing with PBS
124. Assembling with cuvette
125. Putting a certain amount of dye and PBS within cuvettes
126. Packaging as multiple products and storing
Detailed Description of the Invention
The present invention relates to a single-use pathogen detection chip (1) which enables fluorescent molecules having energy in different wavelengths from each other to interact on the same target (pathogen (60)) thus, allows to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET (Forster Resonance Energy Transfer) event occurred between two molecules, and a production method thereof. Thanks to the
present innovative pathogen detection chip (1), it is possible to detect the presence of pathogen (60) automatically and in a very short time by measuring the change in the emission optical power values without requiring for diagnostic methods applied in laboratory environments.
The present innovative pathogen detection chip (1) generally includes; a chip body (10), a chemical (20) that is coated on mentioned chip body (10), primary probes (30) that are bonded (preferably by covalent bond) to said chip body (10) coated by mentioned chemical (20). Mentioned pathogen detection chip (1) is preferably used by being assembled in a cuvette (50). In addition, secondary probes (40) which are kept in a special solution and used for finalization of the test are included in the same test kit together with said pathogen detection chip (1).
In the described configuration of the invention, the chip body (10) is transparent and preferably made of glass material (cover glass). In a different configuration of the invention, it is possible that the chip body (10) is a non-transparent material too.
For coating on chip body (10), it is possible to use any chemical (20) having a molecular structure that can immobilize at least one of said probes (30, 40) or pathogen (60) by bonding or embedding them on the chip body (10). Preferably any of the silane (a compound containing silicon on one side) molecule organic compounds or THIOL compound can be used. In the preferred embodiment of the invention, THPMP (3-(Trihydroxysilyl)propyl methylphosphonate) chemical (20) which is one of these silane molecule organic compounds, is used to coat the chip body (10). THPMP is a commercial product that is available as a liquid under normal conditions and, has silane molecule at one end and phosphonate molecule at the other end. However, it has not been used in this way before. The reason of using preferably THPMP as the coating chemical (20) in this invention; it enables any biological material to bond (bioconjugation property) to a surface and also have resistance against to adhesion of other proteins to surface of the chip except the proteins related to target pathogen (60). In this way, it is possible to prevent the interaction of undesired materials with the chip while specifically bonding the desired biological material (THPMP increases the selectivity of the chip by showing both the probe bonding property and the property preventing the bonding of foreign substances to the surface). In addition, THPMP allows covalent chemical bonding to be established quickly in a few steps and in high efficiency without requiring an extra molecule. The bonding of antibodies that recognize the pathogen (60) with THPMP is already available in the scientific literature however its usage (usage of THPMP) as a FRET sensor in the pathogen (60) detection is presented for the first time with the present innovative pathogen detection chip (1).
In a preferred embodiment of the invention, THPMP is coated on the transparent chip body (10) by vacuum deposition (vacuum coating) method. For applying the vacuum deposition method, first of
all, THPMP chemical (20) in the liquid form is taken into an isolated container and vacuum is applied to the container. Since the THPMP liquid passes into gas form due to the decrease in the liquid vapor pressure as a result of the vacuum applied, a regular coating is obtained by re-condensing on the chip body (10). It is possible to provide THPMP coating on the chip body (10) within liquid or in any other way however it has been concluded that the coating which has the most homogeneous distribution and the least negative effect on the transparency of chip body (10) is carried out by vacuum deposition method. In the vacuum deposition process, it is possible to reach the best result by optimizing the parameters such as the amount of vacuum, distance of the chip body (10) from the liquid chemical, time etc. Cleaning the chip surface beforehand with piranha solution (Sulfiric acid: Hydrogen peroxide) reveals free hydroxyl groups in the chip body (10) (for example glass) and facilitates the bonding of silane molecules to the chip body (10). Here, alternatively, ultraviolet light and ozone cleaning (UV/ozone) can be applied. In addition, the vacuum deposition method provides a method for coating hundreds of chip bodies (10) at the same time and is a solution that has not been applied before in the literature.
In a preferred embodiment of the invention, after the chip body (10) is coated with the silane molecule organic compound, the coated chip body (10) is kept in vacuum oven at high temperature in order to increase the chemical and mechanical strength of the coating. Thus, by reinforcing the retention of the coating to the chip body (10), it is aimed to obtain a stable coating (which will not be abraded by washing etc. unless we scrape mechanically). The quality of the coating can be evaluated by measuring the contact angle. In the preferred embodiment, the contact angle with the water droplet must be at least 55° in order to proceed with the next step.
As a next step, the primary probes (30) (antibody/protein) which were previously bonded with fluorescent dyes that can be excited at a specific wavelength, are covalently bonded to this coating by using chemical linkers. Since these primary probes (30) are connected with fluorescent dye before excitation (Probes that will be used for pathogen (60) detection are bonded with fluorescent dyes suitable for FRET), a fluorescent coating is obtained on the chip body (10). Since primary probes (30) bonded with fluorescent are covalently bonded to surface coating molecules (preferably THPMP) by using chemical methods and do not directly interact with the chip body (10), the structure of the probes is ensured to remain stable without deterioration. Normally, the three-dimensional structure of any probe such as antibody, protein etc. may be damaged due to their interaction with glass when they adheres to the glass by themselves. In the present innovative solution, the chemical (20) coating on the chip body (10) prevents these probes from interacting with glass (or any other chip body material), thus preserving the three-dimensional structure of antibodies/proteins.
The chip bodies (10) which are coated with chemical (20) and subsequently dyed with primary probes (30) (antibody/protein) bonded with fluorescents, becomes ready for usage. In the preferred embodiment of the invention, it is enabled that the tests are carried out more easily and practically by ensuring the chip body (10) to be assembled preferably with a cylindrical cuvette (50). For this, the pathogen detection chip (1) is attached to the lower side (base) of the cuvette (50) body (51). The cover (52) comprising hole (53) in which swab can enter is attached to the upper side of the cuvette (50) body (51) or can be manufactured as integrally connected with the body (51). Sample taken from the person that will be tested is carried onto the pathogen detection chip (1) within the cuvette (50).
Secondary probes (40) (antibody/protein) kept in solution to be used with the pathogen detection chip (1) in order to detect the presence of the pathogen (60) were previously bonded with fluorescent dye and marked by being excited at a certain wavelength (must be a wavelength different from the wavelength that the primary probe (30) were excited, but again, must be a wavelength that can be excited by the fluorescent emission wavelength of the primary probe (30)).
After the pathogen (60) particles or target molecules in the sample are bonded to the primary probes (30) as a result of placing the sample taken from the person that will be tested onto the pathogen detection chip (1), the secondary probes (40) kept in the solution are poured onto the pathogen detection chip (1). Under normal circumstances, the possibility of that these two probes (30, 40) coexist is low. However, since both probes (30, 40) have affinity (attraction) towards the pathogen (60), these two probes (30, 40) come together side by side in the environment where the pathogen (60) is present since both probes (30, 40) bond to the same pathogen (60), and in this way, the two fluorescent substances in these two probes (30, 40) come together side by side too. Since these fluorescent substances in these probes (30, 40) are marked by being excited at different wavelengths, the energy transfer called as FRET occurs due to the energy difference between them when they come together.
The pathogen detection chip (1) is excited with an excitation energy (70) according to the wavelength of the primary probe (30). For this reason, fluorescent emission is observed from just primary probe (30) when there is no pathogen (60) in the environment (surface of the pathogen detection chip). In the case of that there is pathogen (60) in the environment, fluorescent emission (FRET emission (80)) from the secondary probe (40) is also observed due to energy transfer (FRET) occurred between them. Since the energy transfer takes places only when the pathogen (60) is present in the environment, in this way it is provided both that the presence of the pathogen (60) can be measured very precisely and false positive measurement are eliminated (since this energy transfer does not occur when there is something other than pathogen (60)).
Briefly, first of all, the chip body (10) is coated with silane molecule organic compound, then the pathogen detection chip (1) is obtained by bonding the primary probes (30) with this chemical (20) coating, and finally, as a result of pouring the secondary probes (40) together with swab sample with/without the pathogen (60) to the environment, by means of energy transfer (FRET) occured if there is pathogen (60) in this environment, the presence of the pathogen (60) is detected as thanks to that the chip makes fluorescent emission, namely FRET emission (80), at a wavelength that the chip does not normally emit.
In the present invention, the bonding of fluorescent dyes and probes (30, 40) each other is achieved by using standard techniques. The fluorescent dyes are excited with a laser source. The fluorescent dye bonded to the primary probe (30) and the fluorescent dye bonded to the secondary probe (40) are excited at different wavelengths (namely in different energy levels) from each other. As the wavelength increases, the excitation energy (70) decreases. When the fluorescent dyes are excited at one wavelength, they emit at lower energy wavelength.
Fluorescent dyes are much smaller and simpler molecules than probes (antibodies). Since the fluorescents have a chemical affinity towards probes, they can be bonded to probes. By bonding the dyed and marked-by-being-excited primary probes (30) covalently to the chip body (10) coated with silane molecule organic compound, it is provided that the three-dimensional structure of the probes is protected. Probes normally do not have emitting property, it is provided these probes to gain/have this property by being bonded with fluorescent molecules. Antibodies (probes) are specific to the pathogen (60), they can recognize pathogens (60) and bond to specific site of these pathogens (60). Briefly, the probes bond to the pathogen (60) when the pathogen (60) is present in the environment.
In the probe selection for the present innovative pathogen detection chip (1), antibodies intended for the pathogen (60) recognition were selected. Considerations when selecting antibodies (Specific to COVID-19):
Being confirmed in that it can be used in the diagnosis of COVID- 19 by the World Health Organization,
Being confirmed in that it can be used in the diagnosis by the manufacturer,
Being different antibodies that can recognize different regions/sides on the outer surface of the virus causing COVID-19 disease, The amount of antibody purchased should be dilutable in small proportions.
In the selection of fluorescent dye, as a criteria, it is desired that the dye is bonded to the probe (antibody/protein) with high efficiency. In the FRET application, it is planned to start working with
appropriate antibody marking kits and after obtaining the most successful result in marking of antibodies, to continue with investigating lower cost methods for bonding the probe and fluorescent dye to each other. Accordingly, it was initially preferred to use a FRET pair (two dyes) which can be excited with the ultraviolet/blue and has a FRET emission wavelength in blue/green.
Although the system has been optimized appropriate for antibody/protein probes, nucleic acids (DNA/RNA oligomers or aptamers) designed and produced as complementary of pathogen (60) nucleic acids can be used similarly as probe by being bonded to FRET pairs. In this case, the primary nucleic acid bonded to first FRET dye will be covalently bonded to the surface and the secondary nucleic acid bonded to second FRET dye will be used with the sample. If there are pathogenic nucleic acids in the environment that can be detected by the nucleic acid probes, the FRET dye pair will come together and so FRET emission (80) will occur.
The production steps of the present innovative pathogen detection chip (1) are given in more detailed below. Aim of many of these steps is regarding improvement of the quality of the chemical (20) coating and dyeing process (bonding of primary probes (30) onto the coating). For this reason, such steps aiming to increase the quality of coating and dyeing processes are not mandatory for the production of the present innovative pathogen detection chip (1) and, they can be applied with different methods. Thanks to these steps aiming to increase the quality, it is ensured that the sensitivity of the present innovative pathogen detection chip (1) in pathogen (60) detection is increased.
The production method of the pathogen detection chip (1) consists of 7 steps. These are as follows:
Preliminary preparation phase
Surface cleaning and abrasion processes
Coating and oven curing processes
Control process
Surface functionalization process
Dye coating and cuvette assembly process
Packaging and storing process
Processes in the preliminary preparation phase (without order) are: MES preparation (100), PBS preparation (101), piranha preparation (102), alconox preparation (103), EDC/MES preparation (104), preparation of dye-probe complexes (105) and, positioning chip bodies (10) (namely slides) within holder (106).
Surface cleaning and abrasion processes are respectively: cleaning of the chip body (10) with alconox (107), cleaning the chip body (10) with acetone (108), cleaning the chip body (10) with ethanol (109), rinsing the chip body (10) with distilled water (110), applying first drying operation with nitrogen (111), continuing with piranha process (112), re-rinsing the chip body (10) with distilled water (113), applying second drying operation with nitrogen (114). The chip bodies (10) (for example: glass slides) that will be used in measurement are cleaned with the surface cleaning and abrasion processes by using chemical or physical methods. The aim here is to reveal the hydroxyl groups which allow the chemical molecules to bond and, hidden under a thin carbon layer formed by the powders around under normal conditions.
Coating and oven processes comprise respectively the following steps: coating the chip body (10) whose surface cleaning and abrasion processes are completed, with chemical (preferably silane molecule organic compound) (115) and then, oven curing (116) the coated chip body (10). For coating, THPMP chemical (20) is preferred due to both its bioconjugation and non-specific interaction resistance properties, and organic coating is made by bringing it to the gas phase in vacuum oven. For the coating process, vacuum coating technique is applied by using silanization method (chemical compounds (20) with reactive groups are bonded to glass surfaces by a method called silanization). This process is carried out under vacuum. It is aimed that the chemical and mechanical properties of the coating are increased by applying heat treatment with the oven curing (116) process which takes place after the coating process (115) of the chip body (10) with chemical, thus making it more durable. The oven curing (116) process is preferably carried out as vacuum oven curing.
Control Process comprises respectively the following steps: measuring contact angle (117) of the chip body (10) whose coating and oven curing processes are completed, controlling whether the obtained contact angle is proper or not (118), continuing with next step if the contact angle value is proper, if the contact angle is not proper (119) extirpating the chip body (10) and starting with new one (119). Preferably, the angle value of 55° and above are considered appropriate while the values under it are considered inappropriate.
Surface functionalization process comprises respectively the following steps: agitation of the chip body (10) whose contact angle was determined as appropriate in the control process with EDC/MES (120), washing with MES (preferably with dripping MES) (121). EDC acts as a mediator for bonding of antibody to THPMP in the next dye coating process. MES is a buffer solution with a constant pH value, in which this reaction takes place with high efficiency.
Dye coating and cuvette assembly process comprises respectively the following steps: coating of the chip body (10) exiting from the surface functionalization process with dye and adding PBS (122),
washing with PBS (123) in order to remove surface waste (preferably with dripping PBS) (PBS is a salt and buffer solution which has a pH of 7.4 and is similar to physiological conditions and in which three-dimensional structures of the antibodies remain without deterioration), assembling with cuvette (124) (preferably sealed system), putting a certain amount of dye and PBS within cuvettes (125). Before all this dye coating and cuvette assembly process, in the step of preparation of dye-probe complexes (105) in the preliminary preparation phase, the probes that will be used for pathogen (60) detection are combined with fluorescent dyes suitable for FRET. The above mentioned dye coating process is carried out by bonding the (preferably THPMP) primary probes (30) which are bonded with fluorescent and excited, to the mediators on the coating on the chip body (10) covalently by using chemical methods. Thus, the single-use pathogen detection chip (1) is obtained. In order to make it more useful (for example: kit), it is assembled with the cuvette (50)
In the packaging and storing process, the final products that become pathogen detection chip (1) or kit in the previous step are preferably packaged as multiple products and stored (126).
Although the structure, production steps and pathogen (60) detection way of the present innovative pathogen detection chip (1) are within the scope of the details described above, there are also different alternative embodiment for these.
In the preferred embodiment described in the specification, the primary probe (30) is bonded to chemical (20) coated surface while the secondary probe (40) is free (not bonded to the chemical (20) coated surface). The presence of the pathogen (60) is detected as a result of FRET emission (80) occurred due to the interaction of both probes (30, 40) with the target pathogen (60) that is free. The representative view regarding this event is given in Figure 2b.
In an alternative embodiment of the invention, the primary probe (30) and the secondary probe (40) are bonded to the chemical (20) coated surface. The presence of the pathogen (60) is detected as a result of FRET emission (80) occurred due to the interaction of the free target pathogen (60) with both probes (30, 40). The representative view regarding this event is given in Figure 3b.
In another alternative embodiment of the invention, the target pathogen (60) is bonded to the chemical (20) coated surface and probes (30, 40) are free. The presence of the pathogen (60) is detected as a result of FRET emission (80) occurred due to the interaction of the free primary probe (30) and free secondary probe (40) with the target pathogen (60). The representative view regarding this event is given in Figure 4b.
In another alternative embodiment of the invention, both the primary probe (30) and the secondary probe (40) are free. The presence of the pathogen (60) is detected as a result of FRET emission (80) occurred due to the interaction of both probes (30, 40) with the free target pathogen (60) on the surface (preferable on the chemical (20) coated surface). The representative view regarding this event is given in Figure 5b.
It is possible to make measurement with the present innovative pathogen detection chip (1) in both transmission mode and reflection mode. In the case of measuring in the transmission mode, the chip body (10) should be made of transparent material, preferably glass whereas in the case of measuring in the reflection mode, the chip body (10) should be made of a chemically modifiable material. Finally, although the chemical (20) which has the molecular structure bonding at least one probes (30, 40) or pathogen (60) onto the chip body (10) and is used to coat the chip body (10), is not an indispensable feature for pathogen (60) detection, its usage is very important since it increases the efficiency in pathogen (60) detection.
Claims
CLAIMS A pathogen detection chip (1) which, comprising a chip body (10) and characterized in that; in order to enable fluorescent molecules having energy in different wavelengths from each other to interact on the same target pathogen (60) and thus, to allow to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET event occurred between two molecules; it comprises at least one primary probe (30) bonded with a fluorescent dye that can be marked by being excited at a certain wavelength, at least one secondary probe (40) which is bonded with a fluorescent dye that can be marked by being excited at a different wavelength and, which is used together with mentioned primary probe (30) for resulting the test, a chemical (20) that is coated on the chip body (10) and has the molecular structure that immobilizes at least one of said probes (30, 40) or pathogen (60) by bonding or embedding them on the chip body (10). A pathogen detection chip (1) according to Claim 1 and, wherein mentioned chemical (20) is the silane molecule organic compound or THIOL molecule. A pathogen detection chip (1) according to Claim 2 and, wherein said chemical (20) is THPMP (3-(Trihydroxysilyl)propyl methylphosphonate) which is one of the silane molecule organic compounds. A pathogen detection chip (1) according to one of the preceding claims, wherein mentioned primary probes (30) and secondary probes (40) are antibodies/proteins selected to recognize the pathogen (60) or, nucleic acids (DNA/RNA oligomers or aptamers) that are designed and produced as complementary of pathogen (60) nucleic acids. A pathogen detection chip (1) according to one of the preceding claims, wherein in the case of using optical transmission mode in pathogen detection, the chip body (10) is made of transparent material, preferably made of glass. A pathogen detection chip (1) according to one of the claims 1-4, wherein in the case of using optical reflection mode in pathogen detection, the chip body (10) is made of a chemically modifiable material.
7. A pathogen detection chip (1) according to one of the preceding claims, wherein the pathogen detection chip (1) is used as being assembled on the bottom of a cuvette (50) in order to allow the tests to be made easier and more practically.
8. A pathogen detection chip (1) according to Claim 7 and, characterized in that; the body (51) of mentioned cuvette (50) is preferably in cylindrical form and on its upper side, it comprises cover (52) including a hole (53) in which swab can enter.
9. A pathogen detection chip (1) production method and, characterized in that; in order to enable fluorescent molecules having energy in different wavelengths from each other to interact on the same target pathogen (60) and thus, to allow to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET event occurred between two molecules; it comprises the following steps: preparation of dye-probe complexes (105) by bonding the primary probe (30) and secondary probe (40) with fluorescent dyes that were marked/can be marked by being excited at different wavelengths, coating (115) of chip body (10) with chemical (20) by using silanization method, preferably, coating dye (122) onto the chemical-coated chip body (10) by bonding of the fluorescent-dye-bonded primary probe (30) onto the chemical-coated chip body (10), thus obtaining pathogen detection chip (1) or coating dye (122) onto the chemical-coated chip body (10) by bonding of the fluorescent-dye- bonded primary and secondary probes (30, 40) onto the chemical-coated chip body (10), thus obtaining pathogen detection chip (1).
10. A pathogen detection chip (1) production method according to Claim 9 and, wherein; vacuum coating technique is applied during the coating process (115) of the chip body (10) with chemical in order to ensure homogeneous distribution of the coating on the chip body (10).
11. A pathogen detection chip (1) production method according to Claim 9 or 10 and, characterized in that; in order to preserve the three-dimensional structure of probes; probes and the chemical (20) coated chip body (10) are bonded to each other by covalent bond in the dye coating process of the chip body (10).
12. A pathogen detection chip (1) production method according to one of the claims 9-11 and, characterized by; comprising following steps:
18 preliminary preparation phase consisting of MES preparation (100), PBS preparation (101), piranha preparation (102), alconox preparation (103), EDC/MES preparation (104), preparation of dye-probe complexes (105) and, positioning chip bodies (10) (namely slides) within holder (106), surface cleaning and abrasion processes consisting of the following steps: preferably cleaning of the chip body (10) with alconox (107), cleaning the chip body (10) with acetone (108), cleaning the chip body (10) with ethanol (109), rinsing the chip body (10) with distilled water (110), applying first drying operation with nitrogen (111), continuing with piranha or UV/ozone process (112), re-rinsing the chip body (10) with distilled water (113), applying second drying operation with nitrogen (114), coating and oven curing processes consisting of the following steps: coating the chip body (10) whose surface cleaning and abrasion processes are completed, with chemical (preferably silane molecule organic compound) (115) and then, preferably oven curing
(116) the coated chip body (10) in order to increase the chemical and mechanical properties of the coating, an optional control process consisting of the following steps: measuring contact angle
(117) of the chip body (10) whose coating and oven curing processes are completed, controlling whether the obtained contact angle is proper or not (118), continuing with next step if the contact angle value is proper, if the contact angle is not proper (119) extirpating the chip body (10) and starting with new one (119), surface functionalization process consisting of the following steps: preferably agitation of the chip body (10) whose contact angle was determined as appropriate in the control process with EDC/MES (120) on a shaker, washing with MES (121), dye coating and cuvette assembly process consisting of the following steps: coating of the chip body (10) exiting from the surface functionalization process with dye and adding PBS (122), preferably washing with PBS (123) in order to remove surface waste, preferably assembling with cuvette (124), putting a certain amount of dye and PBS within cuvettes (125),
19 packaging and storing process in which the final products that become pathogen detection chip (1) or kit in the previous step are preferably packaged as multiple products and stored (126).
13. A pathogen (60) detection method and, characterized by; in order to enable fluorescent molecules having energy in different wavelengths from each other to interact on the same target pathogen (60) and thus, to allow to detect the presence of the microbiological structure by observing the change in the emission optical power values resulting from the FRET event occurred between two molecules; in the case of that the primary probe (30) is bonded to the chemical (20) coated surface and the secondary probe (40) is free, obtaining FRET emission (80) resulting from the interaction of two probes (30, 40) with the free pathogen (60) or, in the case of that the primary probe (30) and the secondary probe (40) are bonded to the chemical (20) coated surface, obtaining FRET emission (80) resulting from the interaction of two probes (30, 40) with the free pathogen (60) or, obtaining FRET emission (80) resulting from the interaction of two free probes (30, 40) with the pathogen (60) that is bonded to the chemical (20) coated surface or, obtaining FRET emission (80) resulting from the interaction of two free probes (30, 40) with the free pathogen (60) preferably on the chemical (20) coated surface.
14. A pathogen (60) detection method according to Claim 13 and characterized by allowing to make measurement in both optical transmission mode and optical reflection mode.
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| Application Number | Priority Date | Filing Date | Title |
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| TR2020/21833A TR202021833A2 (en) | 2020-12-26 | 2020-12-26 | DISPOSABLE PATHOGEN DETECTION CHIP AND A RELATED PRODUCTION METHOD |
| TR2020/21833 | 2020-12-26 |
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| TR (1) | TR202021833A2 (en) |
| WO (1) | WO2022139706A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110312751A1 (en) * | 2010-06-17 | 2011-12-22 | Geneasys Pty Ltd | Microfluidic device for detection of mitochondrial dna via fluorescence modulated by hybridization |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110312751A1 (en) * | 2010-06-17 | 2011-12-22 | Geneasys Pty Ltd | Microfluidic device for detection of mitochondrial dna via fluorescence modulated by hybridization |
Non-Patent Citations (2)
| Title |
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| OZCELIK DAMLA, JAIN AADHAR, STAMBAUGH ALEXANDRA, STOTT MATTHEW A., PARKS JOSHUA W., HAWKINS AARON, SCHMIDT HOLGER: "Scalable Spatial-Spectral Multiplexing of Single-Virus Detection Using Multimode Interference Waveguides", SCIENTIFIC REPORTS, vol. 7, no. 1, 1 December 2017 (2017-12-01), XP055954434, DOI: 10.1038/s41598-017-12487-0 * |
| ZHIYONG PENG, STEVEN A. SOPER, MANEESH R. PINGLE, FRANCIS BARANY, LLOYD M. DAVIS: "Ligase Detection Reaction Generation of Reverse Molecular Beacons for Near Real-Time Analysis of Bacterial Pathogens Using Single-Pair Fluorescence Resonance Energy Transfer and a Cyclic Olefin Copolymer Microfluidic Chip", ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, vol. 82, no. 23, 1 December 2010 (2010-12-01), pages 9727 - 9735, XP055080298, ISSN: 00032700, DOI: 10.1021/ac101843n * |
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