A spectroscopy-based sensing device for detecting infectious diseases from biofluids

Abstract

A sensing device for determining pathogens and / or biomarker molecules from a biofluid comprises a body having an inner compartment for receiving a sample holder (1); a Raman or fluorescence spectroscopy system inside the body for determining Raman spectra of a biofluid sample inside the sample holder (1); a processing unit for determining presence of pathogens and / or biomarker molecules in the biofluid sample according to a classification data / algorithm based on the Raman spectra of a pathogen and / or biomarker molecules captured in a primary and a secondary aptamer structures in the biofluid sample; wherein the sensing device further comprises a motion mechanism (2) for moving the sample holder (1); wherein the processing unit is adapted to control the motion mechanism (2) such that the motion mechanism (2) moves the sample holder (1) to align a sample compartment (1.1) with the Raman or fluorescence spectroscopy system to determine the spectra of the biological fluid sample inside a corresponding sample compartment (1.1).

Description

[0001] A SPECTROSCOPY-BASED SENSING DEVICE FOR DETECTING INFECTIOUS DISEASES FROM BIOFLUIDS

[0002] Field of the Invention

[0003] The present invention relates to a spectroscopy-based, especially a Raman or fluorescence spectroscopy-based sensing device for detecting infectious diseases by capturing pathogens (virus, bacterium, or other disease causing microorganisms) via DNA and RNA aptamers as targeting molecules from biofluids using Raman and fluorescence spectroscopy and spectra classification algorithm.

[0004] Background of the Invention

[0005] Rapid and accurate mass screening method for infectious diseases in clinics and at points of care is an urgent grand challenge worldwide, especially for fighting against COVID- 19 and possible future pandemics and their impacts on public health and economies. In the state of the art, the detection technique for the virus is polymerase chain reaction (PCR) (i.e., using nasal swabs for COVID- 19 diagnosis) collected by medical personnel from symptomatic patients applied to clinics. Since the sample collection procedure and testing require expert medical personnel and rely on a limited number of certified test laboratories, turnaround times may extend to multiple days [1,2]. An important ratio of the cases was reported as asymptomatic and may spread the virus without being detected or isolated [3]. Commercial PCR test sensitivities might be as low as 20% depending on viral load [4]. Emergency Use Authorization COVID-19 tests revealed an unreliably wide range of limits of detection (LoD), spanning more than five orders of magnitude differences. Since each 10-fold increase in the LoD of a COVID- 19 diagnostic test is expected to increase the false negative rate by 13%, developing biosensors with a low LoD is critical [5].

[0006] Moreover, rapid detection of variants became essential for timely response against potentially immune-evading mutations. Therefore, a rapid, accurate, clinically validated, point-of-care mass screening test based on easily collected body fluids must be developed to address PCR’s shortcomings. A high- sensitivity detection platform is needed to prevent the pandemics that we are currently in and that may occur in the future, to obtain detailed analysis by simultaneous determination and genetic analysis of virus / pathogen-related diseases with a single sample, and to diagnose biomarkers that may be important for early diagnosis of diseases. Raman spectroscopy is a powerful analysis method that provides a chemical footprint of the samples and helps to overcome the stated problems [6, 7]. Furthermore, another problem in the clinic is the misdiagnoses that occur due to human errors in the clinics. An artificial-intelligence-powered classification algorithm has been developed to diagnose infectious diseases through an analytical sensing system and paved the way to minimize human errors in the diagnosis of infectious diseases.

[0007] Due to the drawbacks mentioned above of the existing detection methods for virus / pathogen-related infections, there is a need for a sensing device to directly detect virus / pathogen-related infections from biofluids using Raman or fluorescence spectroscopy and the classification data / algorithm.

[0008] Brief Description of the Drawings

[0009] An exemplary embodiment of the present invention is illustrated by way of example in the accompanying drawings to be more easily understood, and uses thereof will be clearer when considered in view of the detailed description, in which reference numbers indicate the same or similar elements and the following figures in which:

[0010] Figure 1 is a schematic representation of the top view of the present invention's the motion mechanism and the sample holder.

[0011] The elements illustrated in the figures are numbered as follows:

[0012] 1. Sample holder

[0013] 1.1. Sample compartment

[0014] 2. Motion mechanism

[0015] 2.1. First rod

[0016] 2.2. Second rods 2.3. First motor drive

[0017] 2.4. Second motor drive

[0018] Detailed Description embodiments of the present invention relate to a sensing device for determining pathogens and / or biomarker molecules from a biofluid comprising a body having an inner compartment for receiving a sample holder (1); a Raman or fluorescence spectroscopy system for measuring spectra (Raman or fluorescence spectra) of a biofluid inside the sample holder (1); a processing unit for determining presence of pathogens and / or biomarker molecules in the biological fluid sample according to a classification data / algorithm based on the spectra of a pathogen and / or biomarker molecules captured in a primary and a secondary aptamer structure in the biofluid sample.

[0019] In the embodiments of the invention, the sensing device determines whether the targeted pathogen exists in the biofluid. With the present invention, it is possible to complete more detailed analyses, such as distinguishing variants of the targeted pathogens or biomarker molecules using DNA or RNA aptamers. Moreover, in one embodiment of the invention, the processing unit is also adapted to determine load or concentration of pathogens and / or biomarker molecules in the biofluid sample. In other words, the processing unit is also adapted to determine the pathogen and / or biomarker molecule load / concentration of the biofluid by measuring the spectral (signal) intensity of the pathogen and / or biomarker molecules captured in the primary and the secondary aptamer structure in the biofluid sample.

[0020] The classification data / algorithm providing a diagnosis may be derived from a computer implemented method such as a machine learning. . In one embodiment of the invention, the classification algorithm comprises three parts: pre-processing unit, a smart decision platform, and a post-processing unit. The pre-processing unit improves the classification algorithm’s performance, removes the noise from data, and identifies the parts of the data that contain helpful information for the classification algorithm. The pre-processing unit uses the dimensionality reduction methods such as principal component analysis (PCA) and linear discriminant analysis (LDA), data scaling techniques, and signal and image filters. The output of the pre-processing unit is the data that will be used as input for the classification algorithm. The classification algorithm involves artificial intelligence-based classification of the pre-processed Raman or fluorescence spectroscopy measurements using a classification algorithm such as a Support Vector Machine (SVM). In another embodiment of the invention, other classification algorithms based on t-distributed stochastic neighbor embedding (T-SNE), K-Nearest Neighbor (k-NN), Random Forest, extreme Gradient Boosting (XG-boost), neural networks may be used. The output of the classification algorithm is used by the post-processing unit that decides whether the pathogen is present in the biofluid sample, according to the analysis.

[0021] In one embodiment of the invention, sample holder (1) comprises multiple sample compartments (1.1), each of which is for receiving different biofluid samples, wherein the sensing device further comprises a motion mechanism (2) for moving the sample holder (1); wherein the processing unit is adapted to control the motion mechanism (2) such that the motion mechanism (2) moves the sample holder (1) to align a sample compartment (1.1) with Raman spectroscopy system to determine the Raman spectra of the biofluid sample inside corresponding sample compartment (1.1). In an alternative of this embodiment, the motion mechanism (2) comprises two first rods (2.1) positioned in parallel, a second rod (2.2) orthogonally connected to the two fist rods wherein at least one connection between the second rod (2.2) and the first rod (2.1) is provided via a first motor drive (2.3) such that the first motor drive (2.3) is provided for moving the second rod (2.2) along at least one part of the first rod (2.1), the second rod (2.2) is connected to a base of the inner compartment via a second motor drive (2.4) such that the second motor drive (2.4) is provided for moving the bottom of the interior compartment along at least one part of the second rod (2.2). The bottom of the interior compartment may be the sample holder (1) itself. With the aid of the motion mechanism (2), detecting pathogens and / or biomarker molecules in a biofluid inside each sample compartment is automatized.

[0022] In one embodiment of the invention, the sample holder (1) is in the form of a well plate.

[0023] In one embodiment of the invention, the Raman spectroscopy system comprises a laser driving system having a laser driver, a laser (such as TO-Can packaged, pigtailed, or butterfly laser diodes or LEDs with a visible or a NIR wavelength), and a collimator for collecting laser beams to decrease beam divergence; a laser line filter (2) for removing the spectrum corresponds to the laser source; achromatic lens couples (3,5,9) to increase the light collection capacity and control the laser spot size; an achromatic lens to focus the laser on the objective lens (7); a sample holder (8); a Notch filter (10) for eliminating elastic (Rayleigh) scattering; a mirror (11); an optical lens (12) to focus the laser inside the spectrometer; a Czerny-Turner spectrometer composed of a slit (13), a grating (14), optical mirrors (15,16), and a detector (17) for determining Raman shifts.

[0024] In one embodiment of the invention, the Raman spectroscopy system is a surface- enhanced Raman spectroscopy system to enhance the Raman signal using the nanoplasmonic effect of metal-coated metasurfaces.

[0025] One embodiment of the invention comprises a non-volatile storage for storing the classification data / algorithm based on the Raman spectra.

[0026] One embodiment of the invention comprises a visual interface for showing the determination of the processing unit (the diagnosis) on whether pathogens and / or biomarker molecules in the biofluid are present One embodiment of the invention comprises a network connection module for sending the determination of the processing unit on whether pathogens and / or biomarker molecules in the biofluid are present to a server. In one embodiment of the invention, the biofluid may be saliva, nasal swab, CSF, blood plasma, blood serum, whole blood, tear, urine, vaginal swab, breast milk, sweat or lymph fluid.

[0027] REFERENCES

[0028] [1]. Yeh, Y. T. et al. A rapid and label-free platform for virus capture and identification from clinical samples. Proc Natl Acad Sci U S A 117, 895-901, doi:10.1073 / pnas.1910113117 (2020).

[0029] [2]. Guo, K. et al. Rapid single-molecule detection of COVID-19 and

[0030] MERS antigens via nanobody-functionalized organic electrochemical transistors. Nature Biomedical Engineering 5, 666-677, doi:10.1038 / s41551-021-00734-9 (2021).

[0031] [3]. Oran, D. P. and Topol, E. J., “Prevalence of asymptomatic SARS-CoV-

[0032] 2 infection: A narrative review,” Annals of internal medicine 173, 362- 367 (Sept. 2020).

[0033] [4]. FDA, “Genetic Variants of SARS-CoV-2 May Lead to False Negative

[0034] Results with Molecular Tests for Detection of SARS-CoV-2 - Letter to Clinical Laboratory Staff and Health Care Providers.” U.S. Food and Drug Administration, 8 January 2021 https: / / tinyurl.com / 2p8zmjnv. (Accessed: 28 December 2021).

[0035] [5]. Mackay, M. J. et al. The COVID- 19 XPRIZE and the need for scalable, fast, and widespread testing. Nature Biotechnology 38, 1021-1024, doi: 10.1038 / s41587-020-0655-4 (2020).

[0036] [6]. Bilgin, Buse, Cenk Yanik, Hulya Toran, and Mehmet Cengiz Onbasli.

[0037] 2021. "Genetic Algorithm- Driven Surface-Enhanced Raman Spectroscopy Substrate Optimization” Nanomaterials 11, no. 11: 2905. https: / / doi.org / 10.3390 / nanol 1112905

[0038] [7]. Hulya Torun, Buse Bilgin, Muslum Ilgu, Cenk Yanik, Numan Batur,

[0039] Suleyman Celik, Meric Ozturk, Ozlem Dogan, Onder Ergonul, Ihsan Solaroglu, Fusun Can and Mehmet Cengiz Onbasli. “Machine learning detects SARS-CoV-2 and variants rapidly on DNA aptamer metasurfaces” medRxiv 2021.08.07.21261749;

Claims

CLAIMS1. A sensing device for determining pathogens and / or biomarker molecules from a biofluid comprising a body having an inner compartment for receiving a biofluid sample holder (1); a Raman or a fluorescence spectroscopy system for measuring a spectra of a biofluid sample inside the sample holder (1); a processing unit for determining presence of pathogens and / or biomarker molecules in the biological fluid sample according to a classification data / algorithm based on the spectra of a pathogen and / or biomarker molecules captured in a primary and a secondary aptamer structure in the biofluid sample.

2. The sensing device according to claim 1, wherein the processing unit is adapted to determine the load or concentration of pathogens and / or biomarker molecules in the biological fluid sample by measuring the spectral intensity of the pathogen and / or biomarker molecules captured in the primary and the secondary aptamer structure in the biofluid sample.

3. The sensing device, according to claim 1 or claim 2, wherein sample holder (1) comprises multiple sample compartments (1.1), each of which is for receiving different bio-fluid sample, wherein the sensing device further comprises a motion mechanism (2) for moving the sample holder (1); wherein the processing unit is adapted to control the motion mechanism (2) such that the motion mechanism (2) moves the sample holder (1) to align a sample compartment (1.1) with the Raman or fluorescence spectroscopy system to determine the spectra of the biological fluid sample inside corresponding sample compartment (1.1).

4. The sensing device, according to claim 3, wherein the motion mechanism (2) comprises two first rods (2.1) positioned in parallel, a second rod (2.2) orthogonally connected to the two fist rods wherein at least one connection between the second rod (2.2) and the first rod (2.1) is provided via a first motor (2.3) drive such that the first motor drive (2.3) is provided for moving the second rod (2.2) along at least one part of the first rod (2.1), the second rod (2.2) is connected to a base of the inner compartment via a second motor drive (2.4) such that the second motor drive (2.4) is provided for moving the bottom of the interior compartment along at least one part of the second rod (2.2).

5. The sensing device, according to claim 3 or claim 4, wherein the sample holder (1) is in the form of a well plate.

6. The sensing device, according to any of the preceding claims, wherein the Raman spectroscopy system comprises a laser driving system having a laser driver; a laser; a collimator for collecting laser beams to decrease beam divergence; a laser line filter for removing the spectrum corresponds to the laser source; achromatic lens couples to increase the light collection capacity and control the laser spot size; an achromatic lens to focus the laser on the objective lens (7); a sample holder (8); a Notch filter (10) for eliminating elastic scattering; a mirror (11); an optical lens (12) to focus the laser inside the spectrometer; a Czerny-Turner spectrometer composed of a slit (13), a grating (14), optical mirrors (15,16), and a detector (17) for determining Raman shifts.

7. The sensing device, according to any of the preceding claims, wherein the Raman spectroscopy system is a surface-enhanced Raman spectroscopy system.

8. The sensing device, according to any of the preceding claims, comprises nonvolatile storage for storing the classification data / algorithm based on the spectra.

9. The sensing device, according to any of the preceding claims, comprises a visual interface for showing the determination of the processing unit on whether viral infectious pathogens and / or biomarker molecules in the biological fluid are present.

10. The sensing device, according to any of the preceding claims, comprises a network connection module for sending the determination of the processing unit on whether viral infectious pathogens and / or biomarker molecules in the biological fluid are present to a server.

11. The sensing device, according to any of the preceding claims, wherein the biological fluid is saliva, nasal swab, CSF, blood plasma, blood serum, whole blood, tear, urine, vaginal swab, breast milk, sweat, or lymph fluid.

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