WO2022139707A1 - A specialized biosensor device and pathogen detection method for fret technique - Google Patents

A specialized biosensor device and pathogen detection method for fret technique Download PDF

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Publication number
WO2022139707A1
WO2022139707A1 PCT/TR2020/051424 TR2020051424W WO2022139707A1 WO 2022139707 A1 WO2022139707 A1 WO 2022139707A1 TR 2020051424 W TR2020051424 W TR 2020051424W WO 2022139707 A1 WO2022139707 A1 WO 2022139707A1
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Prior art keywords
biosensor device
radiation
fret
pathogen detection
detection chip
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PCT/TR2020/051424
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French (fr)
Inventor
Ali Aytac SEYMEN
Erol OZGUR
Bulend Ortac
Erkan TUNCAY
Original Assignee
Bilkent Holding A.S
E-A Teknoloji Biyomedikal Cihazlar Dijital Ve Optik Sistemler Nanoteknoloji Arastirma Gelistirme Ithalat Ihracat Taahhut Sanayi Ve Ticaret Limited Sirketi
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Publication of WO2022139707A1 publication Critical patent/WO2022139707A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • the invention relates to a biosensor device that allows the detection of pathogens based on optics specialized for the FRET (Forster Resonance Energy Transfer) technique.
  • FRET Form Resonance Energy Transfer
  • the present invention is about a method that dynamically receives the FRET signal from a disposable (single-use) pathogen detection chip which is specially developed for biosensor device that allows fluorescence molecules with different wavelengths of energy to come together in the same target (pathogen), thus, that allows deciding to the actual existence of the microbiological structure by looking at the change in emission optical power values resulting from the FRET event between two molecules, and by this means enables pathogens to be detected in a short time as 10 seconds.
  • Pathogens are biological entities that can multiply in living cells and cause infection.
  • the majority of pathogens, for example, viruses, are too small to be viewed with an ordinary microscope.
  • the size of the pathogens is usually measured in nanometers. If an influenza A virus is examined, it is understood that it is 80-120 nm in size. Viruses of many types infect all life forms such as humans, plants, bacteria, and algae, causing various diseases. For this reason, it is essential to detect pathogens, especially viruses, sensitively and selectively.
  • Covid-19 coronavirus
  • Covid-19 virus which causes symptoms similar to upper and lower respiratory tract infections such as fever, cough, and sore throat, should be distinguished quickly and practically.
  • Fast detection of the Covid-19 virus is vital for the application of appropriate treatment methods to the patient.
  • a virus test is required before starting work or traveling. Detection methods that give fast and accurate results should be applied because they are taken depending on the said test's results.
  • an activity and inhibitor detection method for the protease of SARS coronavirus 3CL is mentioned. According to this; The formation of the fluorogenic protease substrate of SARS coronavirus 3CL, mixing the substrate with the reaction buffer liquid, and adding 3CL protease to activate in one condition is provided. By detecting the diversity of the SARS coronavirus 3CL protease's fluorescence value, in vitro activity determination provides to be is completed. The 3CL protease and its micro-molecular compound are incubated, waiting for detection in some cases, and mixed with the fluorogenic substrate.
  • the micro molecular compound activity added with 3CL protease is determined. By comparing the two values, the activity inhibition rate of the micro molecular compound with the 3CL protease is calculated. Therefore, it is not mentioned as a method that enables the FRET signal to be dynamically received by a pathogen detection chip developed specifically for a biosensor and thus detecting pathogens in 10 seconds.
  • the aim of this invention is to present a biosensor device that allows the detection of pathogens based on optics specialized for the FRET (Forster Resonance Energy Transfer) technique.
  • Another aim of the invention is to provide a method that enables the FRET signal to be dynamically received over a pathogen detection chip developed specifically for a biosensor device, and thus pathogens can be detected in as little as 10 seconds.
  • Another aim of the invention is to present a biosensor device specialized for FRET technique, which has a compact design and can perform dynamic measurements.
  • the invention presents a system that comprises; a screen that allows the operation result of the biosensor device to be shown, an optic fiber paired with a laser diode, a wavelength selective dichroic mirror that directs laser light from an optic fiber to a pathogen detection chip, a pathogen detection chip specially designed for a biosensor device, a lens that focuses laser light emanating from an optic fiber on the surface of the sample, a collimation system that allows laser light emanating from an optic fiber to be transformed into plane waves, a PMT sensor surface used to detect light, a filter that allows wavelengths other than FRET radiation to be absorbed, a cuvette in which a pathogen detection chip is mounted and a cuvette cover for a cuvette.
  • the invention is a biosensor device that enables the detection of pathogens, and comprises the following steps: using a specially designed collimation system to transform laser light emanating from an optic fiber into a plane wave, using a wavelength selective mirror to direct laser light from an optic fiber to a pathogen detection chip, the lens in front of said pathogen detection chip focuses the laser light emanating from the optic fiber to the sample surface, the part of the fluorescence radiation generated in the pathogen detection chip passes through the path of the illumination light from the optic fiber and collects in the lens, the wavelength of fluorescence radiation unreflecting in the selective mirror and passing through it reaches the PMT sensor surface, the filter in front of PMT sensor surface absorbs external wavelengths of FRET radiation, the said biosensor device ensures that the presence of pathogens is detected through tracking the change of FRET signals over time in order to perform a dynamic measurement.
  • the invention is a pathogen detection method related to a biosensor device that allows the detection of pathogens and comprises the following steps; placing said pathogen detection chip in the biosensor device, the biosensor device is calibrated in order to prevent errors that may occur due to technical reasons, taking data by turning on the laser light emanating from the optic fiber related to the biosensor device and taking the average of the said data, starting the test by placing a sample taken from a patient on the pathogen detection chip, turning on the laser light emanating from the optic fiber related to the biosensor device and starting to collect data from the pathogen detection chip, comparing the data collected from the said pathogen detection chip with the average data of the sample taken from the patient before insertion into the pathogen detection chip, if the resulting FRET radiation increase is above a specific value, the result is positive, if the resulting FRET radiation increase is below a specific value, the result is negative.
  • figure 1 the biosensor device's views and optical system components in figure 1a, figure 1b, figure 1c and figure 1d are shown.
  • the biosensor device performs the calibration process and the device determines this value as the initial value after the calibration process
  • the increase in FRET radiation detected in the biosensor device after the laser light emanating from the optic fiber (20) is turned on, the value taken in the first stage corresponds to the increase in a predetermined value range according to the initial value, the result is shown as positive
  • the biosensor device In the data obtained by the biosensor device, if the increase of FRET radiation continues from the first stage, the biosensor device continues to receive data at repetitive times for 1 minute and if the increase is not stabilized, asking to repeat the test through the biosensor device screen
  • the biosensor device collects data with repetitive periods of 1 minute in order to prevent any error that may occur if the increase in FRET radiation is between the value close to the threshold value in the first stage
  • the biosensor device prompts the sample to be retaken on the screen in case of an increase in the critical value and above the FRET radiation to prevent false-positive results
  • the present invention relates to a biosensor device (10) that allows the detection of pathogens based on optics customized for the FRET (Forster Resonance Energy Transfer) technique.
  • the present invention is about a method that dynamically receives the FRET signal (radiation) from a disposable pathogen detection chip (40) which is specially developed for biosensor device that allows fluorescence molecules with different wavelengths of energy to come together in the same target (pathogen), thus, that allows deciding to the actual existence of the microbiological structure by looking at the change in emission optical power values resulting from the FRET event between two molecules, and by this means enables pathogens to be detected in a short time as 10 seconds.
  • the biosensor device (10) ensures that it is detected by receiving the FRET signal if there is a pathogen in the environment.
  • a primary probe that is excited by light of a specific wavelength, such as ultraviolet, and emits blue emission is bound to the pathogen detection chip (40).
  • Said solution contains a secondary probe that can be excited in blue, and the wavelength of the FRET radiation is green.
  • Said primary probes and secondary probes are nucleic acids (DNA I RNA oligomers or aptamers) that have been designed and produced as conjugates of pathogen nucleic acids or antibodies/proteins selected to recognize the pathogen. Any FRET pair is bound to the FRET donor chip in the system and can be used in the acceptor solution or vice versa.
  • the system includes; a screen (11) that allows the operation result of the biosensor device (10) to be shown, an optic fiber (20) paired with a laser diode, a wavelength selective dichroic mirror (30) that directs laser light from an optic fiber to a pathogen detection chip (40), a pathogen detection chip (40) specially designed for a biosensor device (10), a lens (50) that focuses laser light emanating from an optic fiber (20) on the surface of the sample, a collimation system (60) that allows laser light emanating from an optic fiber (60) to be transformed into plane waves, a PMT sensor surface (70) used to detect light, a filter (71) that allows wavelengths other than FRET radiation to be absorbed, a cuvette (90) in which a pathogen detection chip is mounted and a cuvette cover (91) for a cuvette.
  • the pathogen detection chip (40) is mounted on the bottom of
  • optical system components of the biosensor device (10) of the invention are shown in Figure 2.
  • laser light emanating from an optic fiber (20) of the biosensor device (10) is transformed into a plane waveform using a specially designed collimation system (60).
  • laser light is directed to a pathogen detection chip (40) using a wavelength-selective dichroic mirror (30).
  • the wavelength selective dichroic mirror (30) is a dichroic mirror and reflects values below 450 nm.
  • Lens (50) in front of the pathogen detection chip (40) focuses the light on the sample's upper surface. Generally, the light is first transformed into a plane wave and then focused on the target point.
  • fluorescence radiation with a longer wavelength and equal direction in all directions was modeled in the volume to be formed around the illumination area.
  • Part of the fluorescence light in question passes through the path of the illumination light and collectively in the lens (50).
  • a functional surface (41) located on the upper part of the pathogen detection chip (40)
  • the light is unreflected in the wavelength selective dichroic mirror (30) and passes through the pathogen detection chip (40) and reaches the PMT sensor surface (70).
  • the filter (71) absorbs external wavelengths of FRET radiation.
  • the biosensor device (10) of the present invention uses excitation light radiation detection systems together. In the present invention method, laser light and fluorescence are preferred for excitation light and radiation.
  • Said biosensor device (10) performs dynamic measurement by tracking the change of FRET signals over time.
  • the pathogen detection chip (40) is excited according to the wavelength of a blue-radiating primary probe. Therefore, when there is no pathogen in the environment, blue fluorescence is emitted from the primary probe on the pathogen detection chip (40). In contrast, green fluorescence is observed due to the energy transfer between them in the presence of pathogens.
  • the pathogen's presence can be measured precisely because the energy transfer takes place only when the pathogen is present. Diagnosis takes less than a minute.
  • Figure 3 shows the reflective working version of the optical system of the subject biosensor device. Thanks to a functional surface (41) located at the bottom of the pathogen detection chip (40), the light is reflected in the wavelength selective dichroic mirror (30) and does not pass through the pathogen detection chip (40) but reaches the PMT sensor surface (70). Measurements can be made in both transmission and reflection mode with the biosensor device (10).
  • FIG. 4 An alternative configuration of the optical system related to the biosensor device of the invention is shown in figure 4.
  • the filter (71) instead of the filter (71), there is a second dichroic mirror (31).
  • Said wavelength-selective second dichroic mirror (31) transmits fluorescence radiation which is non-FRET result from pathogen detection chip (40) which has a primary probe to another photodetector (80). FRET radiation is passed.
  • the sensitivity of the biosensor device (10) is increased by making a proportional measurement between fluorescence I FRET radiation.
  • the biosensor device (10) converts the FRET signal generated after the interaction of pathogens with fluorescence molecules into electrical signals.
  • Fluorescence molecules are molecules that are sensitive to light, and when exposed to laser light for a long time, they lose their radiative properties and give negative results.
  • PMT sensor surface (70) placed in the biosensor device (10) converts each incoming emission light into current through the selective filter (71) inside. By changing the PMT power, the amount converted into the current is increased. Increasing this power also increases the background noise that may occur, and the signal changes that will occur will disappear in the noise. For this reason, the biosensor device (10) needs to calibrate each time a pathogen detection chip (40) is placed.
  • the pathogen detection method flow chart regarding the biosensor device is shown.
  • the pathogen detection chip is placed (100) in the biosensor device.
  • the biosensor device performs the calibration process, and after completing the calibration process, the device determines this value as the initial value (105).
  • the biosensor device (10) performs calibration when each pathogen detection chip (40) is placed to prevent errors that may occur due to technical reasons.
  • the power corresponding to the predetermined laser light is adjusted for each pathogen detection chip (40). While the noise is kept at the lowest level in each measurement, it is ensured that the fluorescence molecules can be induced to a fixed value.
  • the biosensor device (10) collects 2000 data per second by turning on the laser light emanating from the optical fiber (20) for 1 second each time, to prevent damage to the fluorescence molecules. After the average of the values collected by the biosensor device (10) and the said value is written into the system memory, the person who will measure through a screen (11) of the biosensor device (10) is asked to place the sample taken from the patient and start the test. Subsequently, the patient's sample is placed on the pathogen detection chip, and the test is started (115).
  • a patient sample interacts with a solution containing a secondary probe that can be excited in blue and has a green wavelength of the FRET radiation before being placed on the pathogen detection chip (40). Then, said pathogen and secondary probe contents are placed on the pathogen detection chip (40). In this way, the primary probe bound on the pathogen detection chip (40) and the fluorescence substance in the secondary probe to which the sample taken from the patient is freely combined is combined. Primary and secondary probes are bound to the same pathogen in the environment.
  • the mentioned primary and secondary probes refer to two probes of different wavelengths and different colors. Therefore, although not limited to blue and green colors, it was stated that the pathogen detection chip (40) and the probe with which the sample is taken from the patient interacted should glow in different colors. After this process, the data is collected when the laser light emanating from the optical fiber (20) is turned on for 1 second at certain stages (120).
  • the data collected are compared with the average data before the patient's sample is placed on the pathogen detection chip (125). Since the said primary and secondary probes are marked and excited at different wavelengths, the energy transfer called FRET occurs due to the energy difference between them when they are brought together.
  • FRET the energy transfer
  • a flow chart about controlling the result obtained regarding the pathogen detection method subject to the invention is shown in Figure 6.
  • the biosensor device continues to receive data at repetitive times for 1 minute, and if it is not fixed, the test is requested to be repeated through the screen of the biosensor device (220). On the screen (11) a text stating that the result could not be determined, repeat the test is shown. If the increase in any time period within the first 1 minute exceeds the threshold value, the result is positive on the screen (225). The biosensor device collects data every repetitive time for 1 minute to prevent the error that may occur if the increase remains between a value close to the threshold value in the first stage (230).

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Abstract

The invention relates to a biosensor device (10) that allows the detection of pathogens based on optics specialized for the FRET technique. It is ensured that the FRET signal is dynamically received over the pathogen detection chip (40) especially developed for a biosensor device (10). Thus, pathogens are detected in a short time, such as 10 seconds. After the sample taken from a patient is mixed with a particular solution, it is dropped on the pathogen detection chip (40) and detected by the biosensor device (10) by receiving the FRET signal if there is a pathogen on the pathogen detection chip (40).

Description

A SPECIALIZED BIOSENSOR DEVICE AND PATHOGEN DETECTION METHOD FOR FRET TECHNIQUE
Technical Field
The invention relates to a biosensor device that allows the detection of pathogens based on optics specialized for the FRET (Forster Resonance Energy Transfer) technique.
More specifically, the present invention; it is about a method that dynamically receives the FRET signal from a disposable (single-use) pathogen detection chip which is specially developed for biosensor device that allows fluorescence molecules with different wavelengths of energy to come together in the same target (pathogen), thus, that allows deciding to the actual existence of the microbiological structure by looking at the change in emission optical power values resulting from the FRET event between two molecules, and by this means enables pathogens to be detected in a short time as 10 seconds.
Prior Art
Pathogens are biological entities that can multiply in living cells and cause infection. The majority of pathogens, for example, viruses, are too small to be viewed with an ordinary microscope. The size of the pathogens is usually measured in nanometers. If an influenza A virus is examined, it is understood that it is 80-120 nm in size. Viruses of many types infect all life forms such as humans, plants, bacteria, and algae, causing various diseases. For this reason, it is essential to detect pathogens, especially viruses, sensitively and selectively.
Significantly, the new coronavirus (Covid-19) needs to be detected as quickly because it affects the world. Covid-19 virus, which causes symptoms similar to upper and lower respiratory tract infections such as fever, cough, and sore throat, should be distinguished quickly and practically. Fast detection of the Covid-19 virus is vital for the application of appropriate treatment methods to the patient. With the introduction of Covid-19 into our lives, a virus test is required before starting work or traveling. Detection methods that give fast and accurate results should be applied because they are taken depending on the said test's results.
With the rapid advancement of technology, it has become easier to detect microorganisms such as viruses and bacteria that are invisible to the eye. Various pathogen detection methods are generally available for PCR (polymerase chain reaction) amplification and fluorescence detection. However, currently used methods do not provide a method that gives fast results such as 10 seconds.
In the patent document no US20080090224, it is mentioned a method and kit for detecting nucleic acids from biological or environmental samples. The method in question; involves amplification of the nucleic acid of interest after conversion into cDNA, if necessary. Then, the amplified nucleic acid is used as a template for further amplification by real-time PCR (RT-PCR) technique. However, an optically based biosensor used for rapid detection of pathogens is not mentioned here.
In the patent document no CN1690691 , an activity and inhibitor detection method for the protease of SARS coronavirus 3CL is mentioned. According to this; The formation of the fluorogenic protease substrate of SARS coronavirus 3CL, mixing the substrate with the reaction buffer liquid, and adding 3CL protease to activate in one condition is provided. By detecting the diversity of the SARS coronavirus 3CL protease's fluorescence value, in vitro activity determination provides to be is completed. The 3CL protease and its micro-molecular compound are incubated, waiting for detection in some cases, and mixed with the fluorogenic substrate. By determining the fluorescence value with the wavelength of the transmitted wave, the micro molecular compound activity added with 3CL protease is determined. By comparing the two values, the activity inhibition rate of the micro molecular compound with the 3CL protease is calculated. Therefore, it is not mentioned as a method that enables the FRET signal to be dynamically received by a pathogen detection chip developed specifically for a biosensor and thus detecting pathogens in 10 seconds.
In the patent document no CN111304366, it is mentioned the Covid- 19 nucleic acid detection method. Accordingly, the method in question; extracting the RNA of a sample to be detected, performing reverse transcription followed by amplifying P using a primer to obtain an amplification product, adding the amplification product to a premix for the reaction determining the reaction product. However, the use of a fluorescently labeled biosensor is not mentioned here.
As a result, the known state of the art is insufficient; dynamically receiving the FRET signal over a pathogen detection chip developed specifically for a biosensor and detecting pathogens in as much as 10 seconds required a solution.
Objectives and Short Description of the Invention
The aim of this invention is to present a biosensor device that allows the detection of pathogens based on optics specialized for the FRET (Forster Resonance Energy Transfer) technique. Another aim of the invention is to provide a method that enables the FRET signal to be dynamically received over a pathogen detection chip developed specifically for a biosensor device, and thus pathogens can be detected in as little as 10 seconds.
Another aim of the invention is to present a biosensor device specialized for FRET technique, which has a compact design and can perform dynamic measurements.
For achieving the aims given above, the invention presents a system that comprises; a screen that allows the operation result of the biosensor device to be shown, an optic fiber paired with a laser diode, a wavelength selective dichroic mirror that directs laser light from an optic fiber to a pathogen detection chip, a pathogen detection chip specially designed for a biosensor device, a lens that focuses laser light emanating from an optic fiber on the surface of the sample, a collimation system that allows laser light emanating from an optic fiber to be transformed into plane waves, a PMT sensor surface used to detect light, a filter that allows wavelengths other than FRET radiation to be absorbed, a cuvette in which a pathogen detection chip is mounted and a cuvette cover for a cuvette.
The invention is a biosensor device that enables the detection of pathogens, and comprises the following steps: using a specially designed collimation system to transform laser light emanating from an optic fiber into a plane wave, using a wavelength selective mirror to direct laser light from an optic fiber to a pathogen detection chip, the lens in front of said pathogen detection chip focuses the laser light emanating from the optic fiber to the sample surface, the part of the fluorescence radiation generated in the pathogen detection chip passes through the path of the illumination light from the optic fiber and collects in the lens, the wavelength of fluorescence radiation unreflecting in the selective mirror and passing through it reaches the PMT sensor surface, the filter in front of PMT sensor surface absorbs external wavelengths of FRET radiation, the said biosensor device ensures that the presence of pathogens is detected through tracking the change of FRET signals over time in order to perform a dynamic measurement. The invention is a pathogen detection method related to a biosensor device that allows the detection of pathogens and comprises the following steps; placing said pathogen detection chip in the biosensor device, the biosensor device is calibrated in order to prevent errors that may occur due to technical reasons, taking data by turning on the laser light emanating from the optic fiber related to the biosensor device and taking the average of the said data, starting the test by placing a sample taken from a patient on the pathogen detection chip, turning on the laser light emanating from the optic fiber related to the biosensor device and starting to collect data from the pathogen detection chip, comparing the data collected from the said pathogen detection chip with the average data of the sample taken from the patient before insertion into the pathogen detection chip, if the resulting FRET radiation increase is above a specific value, the result is positive, if the resulting FRET radiation increase is below a specific value, the result is negative.
Description of the Figures
In figure 1, the biosensor device's views and optical system components in figure 1a, figure 1b, figure 1c and figure 1d are shown.
In figure 2, the optical system components of the biosensor device are shown.
In figure 3, the reflective working version of the optical system related to the biosensor device is shown.
In figure 4, an alternative configuration of the optical system for the subject biosensor device is shown.
In figure 5, the pathogen detection method flow chart regarding the biosensor device is shown.
In figure 6, a flow chart for controlling the result obtained regarding the pathogen detection method is shown. Reference Numbers
10. Biosensor Device
11. Screen
20. Optic Fiber
30. Dichroic Mirror
40. Pathogen Detection Chip
41. Functional Surface
50. Lens
60. Collimation system
70. PMT sensor surface
71. Filter
80. Photodetector
90. Cuvette
91. Cuvette Cover
100. Placing said pathogen detection chip in the biosensor device,
105. The biosensor device performs the calibration process and the device determines this value as the initial value after the calibration process,
110. Taking data by turning on the laser light emanating from the optic fiber related to the biosensor device and taking the average of the said data,
115. Placing a sample taken from a patient on the pathogen detection chip starting the test,
120. Turning on the laser light emanating from the optic fiber related to the biosensor device and starting to collect data from the pathogen detection chip,
125. Comparing the data collected from the said pathogen detection chip with the average data of the sample taken from the patient before insertion into the pathogen detection chip,
130. If the resulting FRET radiation increase is above a specific value, the result is positive
135. If the resulting FRET radiation increase is below a specific value, the result is negative
200. If the increase in FRET radiation detected in the biosensor device, after the laser light emanating from the optic fiber (20) is turned on, the value taken in the first stage corresponds to the increase in a predetermined value range according to the initial value, the result is shown as positive
205. Re-acquisition of data by the biosensor device in the second stage to confirm the positive result obtained by the biosensor device
210. If the increase of FRET radiation remains constant or decreases in the second stage, the result will be shown as positive on the screen
215. Comparison of the data collected in the biosensor device within the specified periods with the previous data
220. In the data obtained by the biosensor device, if the increase of FRET radiation continues from the first stage, the biosensor device continues to receive data at repetitive times for 1 minute and if the increase is not stabilized, asking to repeat the test through the biosensor device screen
225. If the increase of FRET radiation exceeds the threshold value at any time within the first 1 minute, the result will be shown as positive on the screen
230. The biosensor device collects data with repetitive periods of 1 minute in order to prevent any error that may occur if the increase in FRET radiation is between the value close to the threshold value in the first stage
235. At the end of 1 minute, if the increase of FRET radiation remains below the threshold value, the request to repeat the test through the biosensor device screen
240. If there is a decrease in FRET radiation in the first stage, the result will be shown as negative on the screen
245. The biosensor device prompts the sample to be retaken on the screen in case of an increase in the critical value and above the FRET radiation to prevent false-positive results
Detailed Description of the Invention
The present invention relates to a biosensor device (10) that allows the detection of pathogens based on optics customized for the FRET (Forster Resonance Energy Transfer) technique. The present invention; it is about a method that dynamically receives the FRET signal (radiation) from a disposable pathogen detection chip (40) which is specially developed for biosensor device that allows fluorescence molecules with different wavelengths of energy to come together in the same target (pathogen), thus, that allows deciding to the actual existence of the microbiological structure by looking at the change in emission optical power values resulting from the FRET event between two molecules, and by this means enables pathogens to be detected in a short time as 10 seconds.
After the sample taken from a patient is mixed with a particular solution, it is placed on the pathogen detection chip (40). The biosensor device (10) ensures that it is detected by receiving the FRET signal if there is a pathogen in the environment. A primary probe that is excited by light of a specific wavelength, such as ultraviolet, and emits blue emission is bound to the pathogen detection chip (40). Said solution contains a secondary probe that can be excited in blue, and the wavelength of the FRET radiation is green. Said primary probes and secondary probes are nucleic acids (DNA I RNA oligomers or aptamers) that have been designed and produced as conjugates of pathogen nucleic acids or antibodies/proteins selected to recognize the pathogen. Any FRET pair is bound to the FRET donor chip in the system and can be used in the acceptor solution or vice versa.
The views of the biosensor device (10) of the invention from different angles and the optical system components are shown in Figure 1. As seen in the figure 1 , the system includes; a screen (11) that allows the operation result of the biosensor device (10) to be shown, an optic fiber (20) paired with a laser diode, a wavelength selective dichroic mirror (30) that directs laser light from an optic fiber to a pathogen detection chip (40), a pathogen detection chip (40) specially designed for a biosensor device (10), a lens (50) that focuses laser light emanating from an optic fiber (20) on the surface of the sample, a collimation system (60) that allows laser light emanating from an optic fiber (60) to be transformed into plane waves, a PMT sensor surface (70) used to detect light, a filter (71) that allows wavelengths other than FRET radiation to be absorbed, a cuvette (90) in which a pathogen detection chip is mounted and a cuvette cover (91) for a cuvette. The pathogen detection chip (40) is mounted on the bottom of a cuvette (90). The cuvette (90) is preferably cylindrical and includes a cuvette cover (91) with a hole in its upper side through which a test rod can enter.
The optical system components of the biosensor device (10) of the invention are shown in Figure 2. In the present invention, laser light emanating from an optic fiber (20) of the biosensor device (10) is transformed into a plane waveform using a specially designed collimation system (60). Subsequently, laser light is directed to a pathogen detection chip (40) using a wavelength-selective dichroic mirror (30). The wavelength selective dichroic mirror (30) is a dichroic mirror and reflects values below 450 nm. Lens (50) in front of the pathogen detection chip (40) focuses the light on the sample's upper surface. Generally, the light is first transformed into a plane wave and then focused on the target point. After the sample taken from the patient was illuminated with laser light through optic fiber (20), fluorescence radiation with a longer wavelength and equal direction in all directions was modeled in the volume to be formed around the illumination area. Part of the fluorescence light in question passes through the path of the illumination light and collectively in the lens (50). Thanks to a functional surface (41) located on the upper part of the pathogen detection chip (40), the light is unreflected in the wavelength selective dichroic mirror (30) and passes through the pathogen detection chip (40) and reaches the PMT sensor surface (70). The filter (71) absorbs external wavelengths of FRET radiation. The biosensor device (10) of the present invention uses excitation light radiation detection systems together. In the present invention method, laser light and fluorescence are preferred for excitation light and radiation. Said biosensor device (10) performs dynamic measurement by tracking the change of FRET signals over time. The pathogen detection chip (40) is excited according to the wavelength of a blue-radiating primary probe. Therefore, when there is no pathogen in the environment, blue fluorescence is emitted from the primary probe on the pathogen detection chip (40). In contrast, green fluorescence is observed due to the energy transfer between them in the presence of pathogens. The pathogen's presence can be measured precisely because the energy transfer takes place only when the pathogen is present. Diagnosis takes less than a minute.
Figure 3 shows the reflective working version of the optical system of the subject biosensor device. Thanks to a functional surface (41) located at the bottom of the pathogen detection chip (40), the light is reflected in the wavelength selective dichroic mirror (30) and does not pass through the pathogen detection chip (40) but reaches the PMT sensor surface (70). Measurements can be made in both transmission and reflection mode with the biosensor device (10).
An alternative configuration of the optical system related to the biosensor device of the invention is shown in figure 4. Here, instead of the filter (71), there is a second dichroic mirror (31). Said wavelength-selective second dichroic mirror (31) transmits fluorescence radiation which is non-FRET result from pathogen detection chip (40) which has a primary probe to another photodetector (80). FRET radiation is passed. Thus, the sensitivity of the biosensor device (10) is increased by making a proportional measurement between fluorescence I FRET radiation.
The biosensor device (10) converts the FRET signal generated after the interaction of pathogens with fluorescence molecules into electrical signals. Fluorescence molecules are molecules that are sensitive to light, and when exposed to laser light for a long time, they lose their radiative properties and give negative results. PMT sensor surface (70) placed in the biosensor device (10) converts each incoming emission light into current through the selective filter (71) inside. By changing the PMT power, the amount converted into the current is increased. Increasing this power also increases the background noise that may occur, and the signal changes that will occur will disappear in the noise. For this reason, the biosensor device (10) needs to calibrate each time a pathogen detection chip (40) is placed.
In figure 5, the pathogen detection method flow chart regarding the biosensor device is shown. First of all, the pathogen detection chip is placed (100) in the biosensor device. Afterwards, the biosensor device performs the calibration process, and after completing the calibration process, the device determines this value as the initial value (105). The biosensor device (10) performs calibration when each pathogen detection chip (40) is placed to prevent errors that may occur due to technical reasons. The power corresponding to the predetermined laser light is adjusted for each pathogen detection chip (40). While the noise is kept at the lowest level in each measurement, it is ensured that the fluorescence molecules can be induced to a fixed value. Additionally, by turning on the laser light emanating from the optical fiber to the biosensor device, data is received, and the average of the said data is obtained (110). The biosensor device (10) collects 2000 data per second by turning on the laser light emanating from the optical fiber (20) for 1 second each time, to prevent damage to the fluorescence molecules. After the average of the values collected by the biosensor device (10) and the said value is written into the system memory, the person who will measure through a screen (11) of the biosensor device (10) is asked to place the sample taken from the patient and start the test. Subsequently, the patient's sample is placed on the pathogen detection chip, and the test is started (115). A patient sample interacts with a solution containing a secondary probe that can be excited in blue and has a green wavelength of the FRET radiation before being placed on the pathogen detection chip (40). Then, said pathogen and secondary probe contents are placed on the pathogen detection chip (40). In this way, the primary probe bound on the pathogen detection chip (40) and the fluorescence substance in the secondary probe to which the sample taken from the patient is freely combined is combined. Primary and secondary probes are bound to the same pathogen in the environment. The mentioned primary and secondary probes refer to two probes of different wavelengths and different colors. Therefore, although not limited to blue and green colors, it was stated that the pathogen detection chip (40) and the probe with which the sample is taken from the patient interacted should glow in different colors. After this process, the data is collected when the laser light emanating from the optical fiber (20) is turned on for 1 second at certain stages (120).
The data collected are compared with the average data before the patient's sample is placed on the pathogen detection chip (125). Since the said primary and secondary probes are marked and excited at different wavelengths, the energy transfer called FRET occurs due to the energy difference between them when they are brought together. By placing the sample taken from the patient and a secondary probe on the FRET chip, the change of FRET signals over time is followed by the biosensor device (10) and a diagnosis is made regarding the presence of pathogens. If the resulting increase of FRET radiation is above a specific value, the result is considered to be positive (130), if the resulting FRET radiation increase is below a specific value, the result is deemed to be negative (135).
A flow chart about controlling the result obtained regarding the pathogen detection method subject to the invention is shown in Figure 6. The value of the increase in FRET radiation detected in the biosensor device (10) at the first stage after the laser light emanating from the optic fiber (20) is turned on; If it corresponds to an increase in a predetermined value range according to the initial value, the result is shown as positive on the screen (200). This increase occurs in the first stage. However, to confirm the biosensor device's positive result, data is retaken by the biosensor device (10) in the second stage (205). If the increase remains constant or decreases in the second stage, the result is shown as positive in the screen (11) of the biosensor device (10). The biosensor device compares the data collected in each specified period with the previous data (215).
If the fluorescence signal increase (an increase of FRET radiation) continues in the data obtained from the first stage, the biosensor device continues to receive data at repetitive times for 1 minute, and if it is not fixed, the test is requested to be repeated through the screen of the biosensor device (220). On the screen (11) a text stating that the result could not be determined, repeat the test is shown. If the increase in any time period within the first 1 minute exceeds the threshold value, the result is positive on the screen (225). The biosensor device collects data every repetitive time for 1 minute to prevent the error that may occur if the increase remains between a value close to the threshold value in the first stage (230). If the increase in FRET radiation at the end of 1 minute remains below the threshold value, repeating the test is requested through the biosensor device screen (235). It is ensured that a warning stating that the test result could not be determined is shown on the screen (11) of the biosensor device (10), the patient is asked to retake it and repeat the test. Incorrect results generally occur when the sample cannot be taken correctly. However, a decrease in the amount of fluorescence is usually observed in negative results. In the case of fluorescence decrease in the first stage, the result is displayed as negative on the screen (11) of the biosensor device (240). If the signals are above the critical value, it may be caused by the interaction of fluorescence proteins with other molecules or substances resulting from contamination. To prevent the biosensor device's false-positive results, if there is an increase in the critical value and above the FRET radiation, the sample requests to be retaken through the screen (245).

Claims

CLAIMS A biosensor device (10) that allows pathogens to be detected, and characterized by comprising; by means of a screen (11) that allows the operation result of the biosensor device to be shown, an optic fiber (20) paired with a laser diode, a wavelength selective dichroic mirror (30) that directs laser light from an optic fiber (20) to a pathogen detection chip (40), a pathogen detection chip (40) specially designed for a biosensor device (10), a lens (50) that focuses laser light emanating from an optic fiber (20) on the surface of the sample, a collimation system (60) that allows laser light emanating from an optic fiber (20) to be transformed into plane waves, a PMT sensor surface (70) used to detect light and, a filter (71) that allows wavelengths other than FRET radiation to be absorbed; using a specially designed collimation system (60) to transform laser light emanating from an optic fiber (20) into a plane wave, using a wavelength-selective dichroic mirror (30) to direct laser light from an optic fiber (20) to a pathogen detection chip (40), the lens (50) in front of said pathogen detection chip (40) focuses the laser light emanating from the optic fiber (20) to the sample surface, the part of the fluorescence radiation generated in the pathogen detection chip (40) passes through the path of the illumination light from the optic fiber (20) and collects in the lens (50), the wavelength of fluorescence radiation unreflecting in the selective dichroic mirror (30) and passing through it reaches the PMT sensor surface (70), the filter (71) in front of the PMT sensor surface (70) absorbs external wavelengths of FRET radiation, the said biosensor device (10) ensures that the presence of pathogens is detected through tracking the change of FRET signals over time in order to perform a dynamic measurement. A pathogen detection method related to a biosensor device (10) that enables the detection of pathogens, and characterized by comprising the following steps; placing said pathogen detection chip in the biosensor device (100), the biosensor device is calibrated in order to prevent errors that may occur due to technical reasons (105), taking data by turning on the laser light emanating from the optic fiber related to the biosensor device and taking the average of the said data (110), starting the test by placing a sample taken from a patient on the pathogen detection chip (115), turning on the laser light emanating from the optic fiber related to the biosensor device and starting to collect data from the pathogen detection chip (120), comparing the data collected from the said pathogen detection chip with the average data of the sample taken from the patient before insertion into the pathogen detection chip (125), If the resulting FRET radiation increase is above a specific value, the result is positive (130), If the resulting FRET radiation increase is below a specific value, the result is negative (135). A pathogen detection method according to Claim 2, and characterized by comprising the following steps; a primary probe that is excited with a specific wavelength and emits a specific color is connected in said pathogen detection chip (40), the sample taken from a patient freely interacts with a solution containing a secondary probe that can be excited with a specific wavelength and whose FRET radiation wavelength is different from the primary probe, binding a sample from a patient to the primary and secondary probe in the pathogen detection chip (20), excitation of the pathogen detection chip (40) according to the wavelength of a primary probe, If there is no pathogen in the environment, fluorescence radiation of the primary probe on the pathogen detection chip (40) occurs according to the primary probe's wavelength, If there is a pathogen in the environment, different fluorescence radiation occurs according to the secondary probe's wavelength, and this radiation is detected. A pathogen detection method according to Claim 2, and characterized by; comprising the following steps; if the increase in FRET radiation detected in the biosensor device corresponds to an increase in a predetermined value range compared to the initial value in the first stage after the laser light emanating from the optical fiber is turned on, the result is displayed as positive on the screen (200), to confirm the positive result obtained by the biosensor device, data is retaken by the biosensor device in the second stage (205), if the increase in FRET radiation remains constant or decreases in the second stage, the result will be shown as positive on the screen (210), comparing the data collected in the biosensor device with the previous data (215), if the increase in FRET radiation continues in the data obtained by the biosensor device (10) from the first stage, the biosensor device continues to receive data at repetitive times for 1 minute, and if the increase is not stabilized, the test is repeated through the biosensor device screen (220), if the increase in FRET radiation exceeds the threshold value in any time within the first 1 minute, the result will be displayed as positive on the screen (225), the biosensor device (10) collects data in repetitive periods for 1 minute (230) to prevent the error that may occur in case the increase of FRET radiation remains between values close to the threshold value in the first stage, If the increase of FRET radiation remains below the threshold value at the end of 1 minute, asking to repeat the test through the biosensor device screen (235), if there is a decrease in FRET radiation in the first stage, the result is displayed as negative on the screen (240), the biosensor device requests the sample to be retaken through the screen in case of an increase in the critical value and above the FRET radiation to prevent false-positive results (245).
5. A biosensor device (10) that allows pathogens to be detected according to Claim 1 , and characterized in that; said biosensor device (10) uses excitation and radiation detection systems together.
6. A biosensor device (10) according to Claim 5, and characterized in that, it uses excitation and radiation detection systems together and said excitation and radiation detection systems are realized by laser illumination and fluorescence.
7. A biosensor device (10) that allows pathogens to be detected according to Claim 1 , and characterized in that; the biosensor device (10) performs measurements in both pass-through and reflective modes.
8. A biosensor device (10) that allows pathogens to be detected according to Claim 1 and, characterized in that; through a different wavelength selective second dichroic mirror (31) placed in front of the filter (71), it ensures a proportional measurement between fluorescence I FRET radiation by transmitting the non-FRET fluorescence radiation from the primary probe bound pathogen detection chip (40) to a photodetector (80) and passing the FRET radiation.
PCT/TR2020/051424 2020-12-26 2020-12-28 A specialized biosensor device and pathogen detection method for fret technique WO2022139707A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070099204A1 (en) * 2000-03-24 2007-05-03 Isabelle Alexandre Identification and quantification of a plurality of biological (micro)organisms or their components
US20110281775A1 (en) * 2007-07-20 2011-11-17 Eppendorf Ag Detection and/or quantification method of target molecules on a solid support
US20200103412A1 (en) * 2012-04-16 2020-04-02 Commonwealth Scientific And Industrial Research Organisation Methods and systems for detecting an anal yte or classifying a sample

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070099204A1 (en) * 2000-03-24 2007-05-03 Isabelle Alexandre Identification and quantification of a plurality of biological (micro)organisms or their components
US20110281775A1 (en) * 2007-07-20 2011-11-17 Eppendorf Ag Detection and/or quantification method of target molecules on a solid support
US20200103412A1 (en) * 2012-04-16 2020-04-02 Commonwealth Scientific And Industrial Research Organisation Methods and systems for detecting an anal yte or classifying a sample

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