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SYSTEM AND METHOD FOR IDENTIFYING NUCLEOSIDES BASED ON AUTOFLUORESCENCE SPECTRAL PROPERTIES

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SYSTEM AND METHOD FOR IDENTIFYING NUCLEOSIDES BASED ON AUTOFLUORESCENCE SPECTRAL PROPERTIES

ORDINARY APPLICATION

Published

date

Filed on 19 November 2024

Abstract

Embodiments of the present disclosure relate to a system (100) and a method (400) for identifying nucleosides based on autofluorescence spectral properties. The system (100) includes a sample preparation unit, a laser excitation source, a spectrometer, a data processing unit and a comparison unit. The sample preparation unit is configured to prepare a sample solution of nucleosides in Milli-Q water. The laser excitation source is configured to emit a 325 nm pulsed laser to induce autofluorescence in the sample solution. The spectrometer is configured to record the autofluorescence spectra of the nucleosides in the sample solution. The data processing unit is configured to process the recorded spectra to obtain characteristic spectral properties. The comparison unit is configured to compare the characteristic spectral properties of the recorded spectra with reference spectral properties of known nucleosides to identify the nucleosides in the sample solution.

Patent Information

Application ID202441089671
Invention FieldBIO-MEDICAL ENGINEERING
Date of Application19/11/2024
Publication Number47/2024

Inventors

NameAddressCountryNationality
KRISHNA KISHORE MAHATODepartment of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.IndiaIndia
SHIMUL BISWASDepartment of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.IndiaIndia
MRUNMAYEE WANKHEDEDepartment of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.IndiaIndia
JACKSON RODRIGUESDepartment of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.IndiaIndia
SUBHASH CHANDRADepartment of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, 576104, Karnataka, India.IndiaIndia

Applicants

NameAddressCountryNationality
Manipal Academy of Higher EducationMadhav Nagar, Manipal, 576104, Karnataka, India.IndiaIndia

Specification

Description:TECHNICAL FIELD
[0001] The present disclosure generally relates to the field of molecular biology and fluorescence spectroscopy. More particularly, the present disclosure relates to a system and a method for identifying nucleosides based on autofluorescence spectral properties.

BACKGROUND
[0002] Nucleotides are the fundamental building blocks of DNA and RNA. Each nucleotide consists of a nitrogenous base, a sugar moiety, and a phosphate group. The sequence of nucleotides in a DNA strand encodes the genetic information necessary for protein synthesis through transcription and translation. Nucleotides lacking the phosphate group are referred to as nucleosides.
[0003] Current technologies for nucleic acid sequencing, including next-generation sequencing (NGS) and other molecular techniques, are highly accurate but remain prohibitively expensive and resource-intensive for routine clinical applications. This limitation necessitates the development of cost-effective and scalable alternatives.
[0004] Multiple studies describe the use of spectroscopy techniques such as Absorption Spectroscopy, Circular Dichroism (CD) Spectroscopy, Raman Spectroscopy and even Fluorescence Spectroscopy to obtain the individual spectrum for each nucleic acid base, but their implementation in their independent identification has not been documented till date.
[0005] Sanger sequencing was one of the first techniques that was developed for the determination of nucleotide sequences in a DNA strand. It is based on the 'sequencing-by-synthesis' approach, wherein a DNA strand complementary to the target strand is synthesized and used for the sequence determination. Along with dNTPs (deoxynucleotide triphosphate), ddNTPs (Dideoxynucleotide triphosphate) of varying lengths labelled with 4 different fluorescent tags are added to the reaction mixture. Incorporation of dNTPs and ddNTPs results in the formation of multiple ss-DNA molecules of varying lengths. Capillary Electrophoresis is then used to separate these oligos based on their length and the signal from the fluorescently tagged ddNTPs is used to differentiate between each nucleotide added sequentially, and thus the sequence of the original strand can be identified. Even though the Sanger-sequencing technology was revolutionary when it was for its time, because of its low throughput and high costs, it was replaced by second generation technologies, also referred to as 'Next Generation Sequencing' technique.
[0006] The 'gold-standard' tool used at present for nucleoside (and nucleotide) order determination and identification is Next Generation Sequencing (NGS). NGS is capable of simultaneous sequencing of millions of DNA fragments which makes high throughput analysis possible. There are multiple advanced platforms that use NGS for nucleic acid base identification, such as, 454 Pyrosequencing, Illumina, Ion Torrent, SOLiD, DNA nanoball sequencing, PacBio Onso system, PacBio Single-molecule real-time sequencing (SMRT) technology, Helicos single-molecule sequencing, and Nanopore DNA sequencing. Even though NGS is highly reliable and accurate, its employment in the everyday challenges of research does not appear to be practical due to the overall high costs and expertise needed to operate the system and machinery.
[0007] The Nanopore system consists of certain specific 'nano-sensors', that are specialised channels through which the DNA strands pass. The target ds DNA is first denatured, and then the single strand of DNA is directed by certain motor proteins to pass through the pores present, resulting in a disturbance in the channel's electric current. This alteration in the ionic current and is detected by a sensor protein, and is different for each nucleotide, which helps generate a unique signature for each base. In low-complexity sequences, the error rate can increase up to 15%. It has an overall lower read accuracy as compared to short-read sequencers.
[0008] PCR-based methods can be used for the identification of single nucleotides that exist in the form of mutations/SNPs (Single Nucleotide Polymorphisms). The methods can largely be classified in two types - (1) polymorphic or mutant allele-directed specific analysis and (2) melting curve analysis. The first method can be used to detect known SNPs, wherein a primer/probe specific to the mutated nucleotide is used and the recognition of the SNP/mutation is done by confirming the presence or absence of the PCR amplicon. Unlike the mutant allele-directed specific analysis, the melting curve analysis method can be used for the detection of both known and unknown SNPs. The target ds DNA is denatured into ss DNA via heat denaturation and the variation in the fluorescence of dsDNA-binding dye is monitored with respect to the temperature shift. Other post-PCR genotyping techniques such as Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE), Amplification Refractory Mutation System (ARMS), Chemical Mismatch Cleavage (CMC), Restriction Fragment Length Polymorphism (RFLP), Single Strand Conformation Polymorphism (SSCP) are also used for identifying the variations in the nucleic acid sequences. Expensive, time consuming, labour intensive, sensitivity affected by probe. Not suitable for high throughput applications.
[0009] Mass spectrometry, especially MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) is used in the fields of genomics and diagnostics for SNP scoring. To keep the nucleic acids intact during the analysis, the analyte is embedded into a 'matrix'. The analyte-matrix is then volatized resulting in the formation of a particle cloud, containing a mixture of charged ions and uncharged molecules. The ions are separated using their mass/charge ration, and the time required by the particles (time of flight) to reach the detector is measured. The TOF is directly proportional to the mass/charge of the analyte, which can then be used for determining the mass of the nucleic acids. Expensive, complex sample preparation, low mass range.
[0010] DNA microarray technology is a molecular technology that consists of numerous microscopic ss oligonucleotide probes, attached to specific positions on a flat surface, usually made of glass or silicone. Microarrays are one of the most widely used methods for SNP detection. The target DNA is extracted from the sample, followed by amplification by PCR. The amplicons are then labelled using fluorescent dyes (Cy3 or Cy5) and the hybridization of the labelled nucleic acids with the oligonucleotide probes is performed. The DNA microarray plate is then scanned and specific probe/DNA interactions of the sample are measured. These interactions are measured by fluorescence as numerical data, which is then analysed to determine the presence/absence of the SNP. Complex sample preparation, difficulty in detecting SNP in polyploid and complex genomes, cannot genotype unknown SNP locations.
[0011] Clustered regularly interspaced short palindromic repeats (CRISPR)-based methods are widely used for the detection of nucleic acid modifications. In traditional CRISPR systems, CRISPR/Cas12a and CRISPR/Cas13a are used for the detection of nucleic acids. These systems consist of a nuclease, along with a guide RNA and a targeting ssRNA respectively, which aid in the direct detection of unamplified nucleic acids. Recent developments in the area of CRISPR-based systems also describe the incorporation of additional enzymes, proteins, and nanomaterials that can be used to increase and specificity of the detection system, along with lowering the time and expenses needed to perform the same. Confined to in vitro research, complex, expensive.
[0012] The use of external dyes (Cy3 and Cy5) for the 'sequence-identification' of an oligonucleotide has been described. Another study details the use of a conjugated polyelectrolyte, P-C-3, to develop a fluorescence biosensor that evaluates the spectral shape of unknown nucleoside and nucleotide samples for their classification using Machine Learning. The requirement for external agents in nucleic acid base identification increases the overall cost, time, and resources needed for the process.
[0013] To address these limitations, the present invention provides a system and a method for identifying nucleosides based on autofluorescence spectral properties that overcomes the shortcomings of the prior art.

OBJECTS OF THE PRESENT DISCLOSURE
[0014] It is a primary object of the present disclosure to provide a system and a method for identifying nucleosides based on autofluorescence spectral properties.
[0015] It is another object of the present disclosure to develop a system that uses AF properties of nucleosides for their identification to eliminate the need for external fluorescent agents for signal amplification, thereby reducing the overall cost of operation.
[0016] It is yet another object of the present disclosure to provide a method with significantly less labour-intensive due to negligible sample preparation.
[0017] It is yet another object of the present disclosure to provide a system that simultaneous records the nucleic acid base spectra and their analysis can be envisioned, bringing down the total time required to carry out the process.

SUMMARY
[0018] The present disclosure generally relates to the field of molecular biology and fluorescence spectroscopy. More particularly, the present disclosure relates to a system and a method for identifying nucleosides based on autofluorescence spectral properties.
[0019] The primary aspect of the present invention is to design a method and system for identifying nucleosides based on their autofluorescence spectral properties. Nucleosides, which are nucleotides without the phosphate moiety, are the building blocks of DNA and RNA. The identification of these nucleosides is crucial for various applications in molecular biology, microbiology, clinical diagnostics, and pharmaceutical research. Current sequencing technologies, while accurate and reliable, are often expensive and labor-intensive, limiting their widespread use in routine clinical practice. The present invention addresses these limitations by utilizing the intrinsic autofluorescence properties of nucleosides for their identification, thereby eliminating the need for external fluorescent agents and reducing overall costs and complexity.
[0020] In an aspect, a method for identifying nucleosides based on autofluorescence spectral properties is disclosed. The method includes preparing a sample solution of nucleosides in Milli-Q water. The sample solution is excited with a 325 nm pulsed laser to induce autofluorescence. The autofluorescence spectra of the nucleosides in the sample solution is recorded. The recorded spectra are processed to obtain characteristic spectral properties including Emission Maxima (λmax), Full Width Half Maxima (FWHM), and Area Under the Curve (AUC). The method further includes comparing the characteristic spectral properties of the recorded spectra with reference spectral properties of known nucleosides to identify the nucleosides in the sample solution.

BRIEF DESCRIPTION OF DRAWINGS
[0021] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure, and together with the description, serve to explain the principles of the present disclosure.
[0022] FIG. 1 illustrates an exemplary block diagram of the experimental setup of the system for identifying nucleosides based on autofluorescence spectral properties for recording AF spectra of nucleoside and DNA samples, in accordance with an embodiment of the present disclosure.
[0023] FIG. 2 illustrates an exemplary graphical representation of the Normalised autofluorescence spectra of nucleosides (i) Adenosine (ii) Guanosine (iii) Cytidine (iv) Thymidine (v) Uridine and (vi) comparison of all nucleosides together, in accordance with an embodiment of the present disclosure.
[0024] FIG. 3 illustrates an exemplary graphical representation of the Autofluorescence spectrum of (i) Isolated bacterial DNA (ii) Comparison of average AF spectrum of nucleosides with that of bacterial DNA (Normalized) (iii) Comparison of AF spectrum of bacterial DNA with AF spectra of nucleosides (Normalized), in accordance with an embodiment of the present disclosure.
[0025] FIG. 4 illustrates an exemplary flowchart explaining the method for identifying nucleosides based on autofluorescence spectral properties, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION
[0026] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit, and scope of the present disclosure as defined by the appended claims.
[0027] Specific details are given in the following description to provide a thorough understanding of the embodiments. However, it will be understood by one of ordinary skill in the art that the embodiments may be practiced without these specific details. For example, circuits, systems, networks, processes, and other components may be shown as components in block diagram form in order not to obscure the embodiments in unnecessary detail. In other instances, well-known circuits, processes, algorithms, structures, and techniques may be shown without unnecessary detail to avoid obscuring the embodiments.
[0028] Also, it is noted that individual embodiments may be described as a process that is depicted as a flowchart, a flow diagram, a data flow diagram, a structure diagram, or a block diagram. Although a flowchart may describe the operations as a sequential process, many of the operations can be performed in parallel or concurrently. In addition, the order of the operations may be re-arranged. A process is terminated when its operations are completed but could have additional steps not included in a figure. A process may correspond to a method, a function, a procedure, a subroutine, a subprogram, etc. When a process corresponds to a function, its termination can correspond to a return of the function to the calling function or the main function.
[0029] The present disclosure generally relates to the field of molecular biology and fluorescence spectroscopy. More particularly, the present disclosure relates to a system and a method for identifying nucleosides based on autofluorescence spectral properties.
[0030] The present disclosure implements fluorescence spectroscopy to identify individual nucleosides based on their autofluorescence spectral properties (Emission Maxima- max, Full Width Half Maxima (FWHM), Area Under the Curve (AUC)). Autofluorescence spectra of all samples (nucleosides and DNA) dissolved in Mili-Q water were recorded at neutral pH. The DNA was isolated from Klebsiella sp. and the corresponding intrinsic fluorescence spectra were recorded to check for similarities between the isolated DNA sample and the pure nucleoside samples.
[0031] FIG. 1 illustrates an exemplary block diagram of the experimental setup of the system for identifying nucleosides based on autofluorescence spectral properties for recording AF spectra of nucleoside and DNA samples, in accordance with an embodiment of the present disclosure.
[0032] With reference to Fig. 1, the system (100) includes a sample preparation unit, a laser excitation source, a spectrometer, a data processing unit and a comparison unit. The sample preparation unit is configured to prepare a sample solution of nucleosides in Milli-Q water. The laser excitation source is configured to emit a 325 nm pulsed laser to induce autofluorescence in the sample solution. The spectrometer is configured to record the autofluorescence spectra of the nucleosides in the sample solution. The data processing unit is configured to process the recorded spectra to obtain characteristic spectral properties. The comparison unit is configured to compare the characteristic spectral properties of the recorded spectra with reference spectral properties of known nucleosides to identify the nucleosides in the sample solution,
[0033] In an embodiment, the sample preparation unit is configured to dissolve pure nucleoside samples in Milli-Q water to a concentration of 10 mM. The laser excitation source is a Nd-YAG pumped dye laser. The data processing unit is configured to perform smoothening, interpolation, baseline correction, and overall processing of the recorded spectra using software tools such as ThorSpectra and GRAMS-AI. The data processing unit is further configured to visualize the processed data using software tools such as SpectraGryph 1.2 and GraphPad Prism 8.0 and is further configured to normalize the autofluorescence spectra of the nucleosides before processing.
[0034] In an embodiment, the comparison unit is configured to compare the characteristic spectral properties with reference spectral properties stored in a database for identification. The nucleosides identified include but not limited to adenosine, guanosine, cytidine, thymidine, and uridine. The spectrometer is configured to record the autofluorescence spectra at neutral pH. Bacterial DNA is isolated separately using a standard protocol, dissolved in Milli-Q water, and subsequently transferred into a cuvette for recording autofluorescence spectra.
[0035] Materials: Nucleoside samples (Adenosine, Guanosine, Cytosine, Thymidine, Uridine), Milli-Q water, Tris Base, 100% ethanol, EDTA, SDS, sodium acetate. Sample Preparation: Pure nucleoside samples were acquired from Sigma-Aldrich Products. 10 mM stock solutions were prepared for each nucleoside by dissolving the appropriate mass of the pure nucleoside in 2 mL of Milli-Q water. Autofluorescence (AF) Spectroscopy: Nd-YAG (Neodymium-doped Yttrium Aluminium Garnet) pumped dye laser was used as the excitation source and 325 nm was the excitation wavelength used to excite all nucleoside samples throughout the study. AF spectrum of 1.5 mL Milli-Q water was recorded prior to the recording of the sample spectra. The resulting spectrum was treated as the background and subtracted from each of the individual spectra that were recorded for the samples. The smoothening, interpolation, baseline correction, and overall processing of the recorded spectra was done using the software ThorSpectra and GRAMS-AI. The visualization of the obtained data was done using SpectraGryph 1.2 and GraphPad Prism 8.0.
[0036] The main aspects of the present disclosure includes use of autofluorescence signatures of nucleosides for their identification, Characteristic Spectral properties of nucleosides (Emission wavelength, Full Width Half maxima, Area Under the Curve), Application: The method can be used in diagnostic tools, and research applications, and potential integration with machine learning algorithms are possible for automated nucleic acid base identification and Real-time Analysis: The technique can be used for real-time identification, improving the efficiency and speed of nucleotide analysis.
[0037] FIG. 2 illustrates an exemplary graphical representation (200) of the Normalised autofluorescence spectra of nucleosides (i) Adenosine (ii) Guanosine (iii) Cytidine (iv) Thymidine (v) Uridine and (vi) comparison of all nucleosides together, in accordance with an embodiment of the present disclosure.
[0038] In an embodiment, the below results report the characteristic autofluorescence spectral properties of all five nucleosides found in DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid). The max , FWHM, and AUC were seen to remain relatively consistent over multiple recordings and various days for all nucleosides. It can be observed that the 𝜆max, FWHM, and AUC appear to be similar for the pyrimidines (thymidine, cytidine, and uridine) and also for the purines (adenosine and guanosine). The 𝜆max for the pyrimidines is around the 400 nm range whereas the 𝜆max for the purines is above 410 nm. The shape of the peak for the pyrimidines also has a comparatively smaller FWHM, appearing to be sharp in nature, whereas the peak shape for the purines is quite broad, resulting in a larger FWHM. Further development in this area might make it possible to use the unique spectral properties of these nucleic acid bases for their independent autofluorescence properties-based recognition in a target DNA strand. These spectral parameters (Emission Maxima- max, Full Width Half Maxima (FWHM), and Area Under the Curve (AUC)) can also be exploited in the fluorescence-based NGS platforms, and the need for external fluorescent dyes for labelling the nucleic acid bases can potentially be eliminated, making the overall sequencing process more efficient and less expensive.

[0039] The above results represent the autofluorescence spectra of all nucleosides found in DNA (Adenosine, Guanosine, Cytidine, Thymidine). The average spectrum of all individual nucleosides was calculated, in order to compare it with the pure isolated DNA extracted from bacterial colonies of Klebsiella spp. Upon comparison of the normalized spectrum of the pure DNA sample with that of the normalized average spectrum of all nucleosides (except uridine), it was observed that the two spectra were almost exactly overlapping. This strengthens the earlier obtained results regarding the autofluorescence properties of nucleosides.
[0040] FIG. 3 illustrates an exemplary graphical representation (300) of the Autofluorescence spectrum of (i) Isolated bacterial DNA (ii) Comparison of average AF spectrum of nucleosides with that of bacterial DNA (Normalized) (iii) Comparison of AF spectrum of bacterial DNA with AF spectra of nucleosides (Normalized), in accordance with an embodiment of the present disclosure.
[0041] FIG. 4 illustrates an exemplary flowchart explaining the method (400) for identifying nucleosides based on autofluorescence spectral properties, in accordance with an embodiment of the present disclosure.
[0042] With reference to Fig. 2, at step 402, preparing a sample solution of nucleosides in Milli-Q water.
[0043] At step 404, exciting the sample solution with a 325 nm pulsed laser to induce autofluorescence.
[0044] At step 406, recording the autofluorescence spectra of the nucleosides in the sample solution.
[0045] At step 408, processing the recorded spectra to obtain characteristic spectral properties including Emission Maxima (λmax), Full Width Half Maxima (FWHM), and Area Under the Curve (AUC).
[0046] At step 410, comparing the characteristic spectral properties of the recorded spectra with reference spectral properties of known nucleosides to identify the nucleosides in the sample solution.
[0047] In an implementation of an embodiment, a method for identifying nucleosides and determining the sequence of nucleotides in a DNA sample using fluorescence spectroscopy is provided. The Nucleoside Samples are prepared. Individual nucleosides, including adenosine, cytidine, guanosine, and thymidine, were procured in pure form. Each nucleoside was dissolved in 1.5 mL Milli-Q water. The pH of the solution was adjusted to neutral (pH 7) to ensure consistency across all samples. The autofluorescence spectra of the prepared nucleoside solutions were recorded using a fluorescence spectrometer equipped with an excitation source in the UV range (300-400 nm). The emission maxima (λmax), full width at half maxima (FWHM), and area under the curve (AUC) of each nucleoside's emission spectrum were determined and recorded. Distinct spectral properties were observed for each nucleoside, allowing for their identification based on fluorescence signatures. Genomic DNA is isolated from Klebsiella sp. using a standard phenol-chloroform extraction protocol. The isolated DNA was purified and dissolved in Milli-Q water at a concentration of 0.01 M. The solution was adjusted to neutral pH to match the conditions used for nucleoside samples. The autofluorescence spectrum of the DNA sample was recorded under the same conditions used for the nucleoside samples. The λmax, FWHM, and AUC values of the DNA spectrum were analyzed to identify peaks and spectral features that correlated with those of the pure nucleoside samples. The spectral features of the DNA sample were compared to the spectral properties of the nucleosides. Correlations between the DNA spectral peaks and the characteristic fluorescence signatures of individual nucleosides were established. Using the observed spectral matches, the sequence of nucleotides in the DNA sample was inferred. To validate the accuracy of the method, the nucleotide pattern obtained through fluorescence spectral analysis was compared with a fluorescence pattern of the Klebsiella sp. DNA obtained using autofluorescence measurements. The results demonstrated that the fluorescence-based method provided a reliable and cost-effective alternative for nucleotide sequence determination.
[0048] In an exemplary embodiment, an interesting direction where the autofluorescence properties of nucleic acid bases can be pivoted is in their unaided identification. The sequence in which nucleotides are present in a DNA strand determines the type and/or the amount of the particular protein that will be synthesized after undergoing the processes of transcription and translation. Therefore, the sequence of these nucleic acid bases is extremely crucial for the functioning and the very existence of all living organisms. Understandably, the determination of this particular order of nucleotides has numerous applications in a wide variety of fields, ranging from scientific fields such as molecular biology, and microbiology to clinical and pharmaceutical fields as well. Currently, sequencing is the major tool used for the determination of nucleotide order determination. Even though sequencing platforms are highly reliable and accurate, their employment in solving the everyday challenges of research does not appear to be practical due to the high costs and expertise needed to operate the system and machinery. Therefore, there is a scope for the development of a novel technology that can aid in reducing the costs of nucleic acid base identification, thus making the entire process more convenient and accessible for all.
[0049] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are comprised to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
, Claims:1. A system (100) for identifying nucleosides based on autofluorescence spectral properties, the system (100) comprising:
a sample preparation unit configured to prepare a sample solution of nucleosides in Milli-Q water;
a laser excitation source configured to emit a 325 nm pulsed laser to induce autofluorescence in the sample solution;
a spectrometer configured to record the autofluorescence spectra of the nucleosides in the sample solution;
a data processing unit configured to process the recorded spectra to obtain characteristic spectral properties including Emission Maxima (λmax), Full Width Half Maxima (FWHM), and Area Under the Curve (AUC); and
a comparison unit configured to compare the characteristic spectral properties of the recorded spectra with reference spectral properties of known nucleosides to identify the nucleosides in the sample solution.

2. The system (100) as claimed in claim 1, wherein the sample preparation unit is configured to dissolve pure nucleoside samples in Milli-Q water to a concentration of 10 mM.

3. The system (100) as claimed in claim 1, wherein the laser excitation source is a Nd-YAG pumped dye laser.

4. The system (100) as claimed in claim 1, wherein the data processing unit is configured to perform smoothening, interpolation, baseline correction, and overall processing of the recorded spectra using software tools such as ThorSpectra and GRAMS-AI.
5. The system (100) as claimed in claim 1, wherein the data processing unit is further configured to visualize the processed data using software tools such as SpectraGryph 1.2 and GraphPad Prism 8.0 and is further configured to normalize the autofluorescence spectra of the nucleosides before processing.

6. The system (100) as claimed in claim 1, wherein the comparison unit is configured to compare the characteristic spectral properties with reference spectral properties stored in a database for identification.

7. The system (100) as claimed in claim 1, wherein the nucleosides identified include but not limited to adenosine, guanosine, cytidine, thymidine, and uridine.

8. The system (100) as claimed in claim 1, wherein the spectrometer is configured to record the autofluorescence spectra at neutral pH.

9. The system (100) as claimed in claim 1, wherein bacterial DNA is isolated separately using a standard protocol, dissolved in Milli-Q water, and subsequently transferred into a cuvette for recording autofluorescence spectra.

10. A method (400) for identifying nucleosides based on autofluorescence spectral properties, the method (400) comprising the steps:
preparing (402) a sample solution of nucleosides in Milli-Q water;
exciting (404) the sample solution with a 325 nm pulsed laser to induce autofluorescence;
recording (406) the autofluorescence spectra of the nucleosides in the sample solution;
processing (408) the recorded spectra to obtain characteristic spectral properties including Emission Maxima (λmax), Full Width Half Maxima (FWHM), and Area Under the Curve (AUC); and
comparing (410) the characteristic spectral properties of the recorded spectra with reference spectral properties of known nucleosides to identify the nucleosides in the sample solution.

Documents

NameDate
202441089671-Proof of Right [06-12-2024(online)].pdf06/12/2024
202441089671-COMPLETE SPECIFICATION [19-11-2024(online)].pdf19/11/2024
202441089671-DECLARATION OF INVENTORSHIP (FORM 5) [19-11-2024(online)].pdf19/11/2024
202441089671-DRAWINGS [19-11-2024(online)].pdf19/11/2024
202441089671-EDUCATIONAL INSTITUTION(S) [19-11-2024(online)].pdf19/11/2024
202441089671-EVIDENCE FOR REGISTRATION UNDER SSI [19-11-2024(online)].pdf19/11/2024
202441089671-EVIDENCE FOR REGISTRATION UNDER SSI(FORM-28) [19-11-2024(online)].pdf19/11/2024
202441089671-FORM 1 [19-11-2024(online)].pdf19/11/2024
202441089671-FORM FOR SMALL ENTITY(FORM-28) [19-11-2024(online)].pdf19/11/2024
202441089671-FORM-9 [19-11-2024(online)].pdf19/11/2024
202441089671-POWER OF AUTHORITY [19-11-2024(online)].pdf19/11/2024
202441089671-REQUEST FOR EARLY PUBLICATION(FORM-9) [19-11-2024(online)].pdf19/11/2024

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