Use of glyconanomaterial in DNA polymerase

Abstract

Provided is use of a glyconanomaterial for inhibiting a DNA polymerase. The glyconanomaterial is generated by means of dry heating of a saccharide, and the glyconanomaterial comprises a graphene-like nanosheet and the saccharide, wherein the graphene-like nanosheet comprises a carbonization product of at least a part of the saccharide, and the graphene-like nanosheet and the saccharide are combined to form a cross-linked supramolecular structure. The glyconanomaterial can be applied to a hot-start polymerase chain reaction, thereby improving the sensitivity and specificity of the reaction.

Description

Applications of sugar nanomaterials in DNA polymerase Technical Field

[0001] This invention relates to the fields of nanopolymer engineering and polymerase chain reactions, and in particular to sugar nanomaterials and their use in inhibiting DNA polymerase. Background Technology

[0002] Polymerase chain reaction (PCR) is a commonly used technique in molecular biology to amplify specific nucleic acid sequences. Its core technology lies in using DNA polymerase to replicate nucleic acid sequences in vitro, thereby amplifying the target nucleic acid fragment by more than a million times. This allows a small amount of sample to be amplified to an analytical quantity. This technique can be applied to the qualitative, quantitative, sequencing, and detection of nucleic acids, and its application fields are quite extensive.

[0003] Hot-start polymerase chain reaction (HS-PCR) is an improvement on traditional PCR technology, designed to enhance the specificity and sensitivity of amplification. It controls the activity of DNA polymerase to reduce non-specific amplification, such as primer dimers. Before the reaction reaches its optimal operating temperature, DNA polymerase may perform unnecessary amplification at low temperatures, leading to non-specific products and affecting the accuracy of the results. Hot-start PCR prevents non-specific amplification during the preparation phase by inhibiting DNA polymerase activity before the reaction begins, allowing the polymerase to regain activity only after the PCR temperature has been raised to a certain level.

[0004] Currently, hot-start PCR commonly uses antibodies or aptamers as ligands to inhibit polymerase activity. These ligands bind to the active site of DNA polymerase at low temperatures before the PCR reaction begins, inhibiting its activity. Once the PCR temperature is raised to the denaturation temperature, the antibody or aptamer loses its activity, releasing the DNA polymerase and restoring its activity for amplification of the target fragment. However, antibodies and aptamers are only effective at low temperatures and are extremely unstable during PCR heating, failing to maintain their inhibitory properties. Furthermore, they denature at high temperatures, making them unusable during the reaction process. Their preparation is also complex and costly. Therefore, further development of more stable and reversible DNA polymerase inhibitory materials is needed in this field. Summary of the Invention

[0005] Based on the foregoing, this invention provides a carbonized nanomaterial with DNA polymerase inhibitory activity. By binding to DNA polymerase, it achieves the effect of inhibiting polymerase activity and can produce reversible inhibitory properties according to temperature changes. It can effectively improve the specificity and sensitivity of PCR reactions, and is easy to prepare and cost-effective.

[0006] In one aspect, the present invention provides the use of a sugar nanomaterial for inhibiting DNA polymerase, wherein the sugar nanomaterial is generated by dry heating of sugars at 150°C to 300°C, wherein the sugar nanomaterial comprises graphene-like nanosheets and the sugars, wherein the graphene-like nanosheets comprise at least a portion of the carbonized products of the sugars, and the graphene-like nanosheets and the sugars are combined to form a cross-linked supramolecular structure.

[0007] In some specific embodiments, the sugar is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides and their derivatives.

[0008] In some specific embodiments, the sugar is selected from the group consisting of glucose, sucrose, dextrin, sodium alginate, agarose, hydroxyethyl cellulose and xanthan gum.

[0009] In some specific embodiments, the surface of the cross-linked supramolecular structure has functional groups selected from the group consisting of hydroxyl, ester, phenol, carboxyl, and any combination thereof.

[0010] In some specific embodiments, the sugar nanomaterial inhibits the activity of the DNA polymerase by binding to it, and this inhibition is reversible.

[0011] In some specific embodiments, the sugar nanomaterial is used for a thermally initiated polymerase chain reaction, in which the sugar nanomaterial binds to DNA polymerase to inhibit its activity, and releases the DNA polymerase by increasing the reaction temperature, thereby restoring its activity.

[0012] In another aspect, the present invention provides a method for improving the precision of polymerase chain reactions, which involves adding a sugar nanomaterial to the polymerase chain reaction. The sugar nanomaterial is generated by dry heating of sugars at 150°C to 300°C. The sugar nanomaterial comprises graphene-like nanosheets and the sugars, wherein the graphene-like nanosheets contain at least a portion of the carbonized products of the sugars, and the graphene-like nanosheets and the sugars are combined to form a cross-linked supramolecular structure. This precision includes both sensitivity and specificity.

[0013] In some specific embodiments, the sugar nanomaterial inhibits the activity of DNA polymerase by binding it and releases the DNA polymerase by increasing the reaction temperature, thereby restoring its activity.

[0014] In some specific embodiments, the concentration of the sugar nanomaterial is from 1 μg / ml to 2500 μg / ml.

[0015] In some specific embodiments, the polymerase chain reaction includes conventional polymerase chain reaction, hot-start polymerase chain reaction (PCR), capillary electrophoresis polymerase chain reaction (PCR-capillary electrophoresis), real-time polymerase chain reaction (real-time PCR), multiplex polymerase chain reaction (multiplex PCR), or allele-specific polymerase chain reaction (allele-specific PCR).

[0016] The sugar nanomaterials of this invention are prepared by an environmentally friendly, easily scaled-up, and highly reproducible one-pot synthesis method, which is prepared from cost-effective and readily available sugars or sugar precursors without the use of any catalysts or toxic solvents.

[0017] This invention employs carbonization synthesis technology, optimizing sugars through different thermal conversion temperatures, reaction times, and purification conditions. This allows the carbonized products to achieve different carbonization structures and surface functional group distributions depending on the selection of different precursors. The saccharide nanomaterials provided by this invention possess reversible DNA polymerase inhibitory properties. In PCR reactions, through higher temperature stability, they enable PCR hot-start functionality, thereby resolving PCR multiple banding and primer dimer phenomena, lowering the qPCR cycle threshold (Ct), increasing PCR amplification yield, optimizing multiplex PCR efficiency, improving variant detection capabilities, and providing more specific genotyping results, achieving higher accuracy. Attached Figure Description

[0018] Figure 1 is a schematic diagram of the generation of the sugar nanomaterials described in this invention.

[0019] Figure 2 shows the inhibitory effect of sugar nanomaterials generated from different sugar precursors on DNA polymerase.

[0020] Figure 3A shows photographs of sodium alginate after dry heating at 150℃, 200℃, 250℃, and 300℃ (top image) and dissolved in deionized water (bottom image); Figure 3B shows transmission electron microscopy (TEM) images of sodium alginate after dry heating at 150℃, 200℃, 250℃, and 300℃, with scale bars of 1000nm, 2000nm, 250nm, and 500nm respectively; Figure 3C shows high-resolution transmission electron microscopy (HRTEM) images of sodium alginate after dry heating at 150℃, 200℃, 250℃, and 300℃, with a scale bar of 5nm and a mark length of 0.24nm.

[0021] Figure 4 shows the Fourier transform infrared (FT-IR) spectra of the sugar nanomaterials generated by dry heating of sodium alginate at 150℃, 200℃, 250℃ and 300℃, and a table of functional groups corresponding to each absorption peak.

[0022] Figure 5A shows the electrophoresis results of PCR experiments on the sugar nanomaterials generated by dry heating sodium alginate at 150℃, 200℃, 250℃, and 300℃. The target sequence size in the figure is 653bp. Figure 5B shows the electrophoresis results of PCR experiments on the sugar nanomaterials generated by dry heating glucose at 150℃, 180℃, and 210℃. The target fragment size in the figure is 406bp, where the symbol... The position is the length of the target segment.

[0023] Figure 6 shows the effect of sodium alginate dry heating at 250℃ on the relative activity of DNA polymerase at temperatures of 35℃, 45℃, 55℃, 65℃ and 75℃ (*** represents p<0.001).

[0024] Figure 7A shows the electrophoresis diagram of the effect of the sugar nanomaterial generated by dry heating sodium alginate at 250℃ on PCR under different concentrations of DNA template. Lanes 1, 2, 3, 4, and 5 correspond to 30, 3, 0.3, 0.03, and 0 ng of DNA template, respectively. Figure 7B shows the electrophoresis diagram of PCR reactions with different concentrations of sodium alginate nanomaterial generated by dry heating at 250℃. Figure 7C shows the electrophoresis diagram of the effect of adding (right) and not adding (left) the sugar nanomaterial of the present invention on the PCR reaction. Figure 7D shows the electrophoresis diagram comparing the addition of the sugar nanomaterial of the present invention with commercially available hot-start PCR reagents. Lanes 1, 2, 3, and 4 correspond to the sugar nanomaterial of the present invention, DreamTaq DNA polymerase (Thermo Scientific, USA), DreamTaq Hot Start DNA polymerase (Thermo Scientific, USA), and KAPA2G Fast HotStart PCR, respectively. Kit (Roche, Switzerland), where black indicators represent the position of the target sequence length; Figure 7E shows the electrophoresis images of the sugar nanomaterials described in this invention reacting with commercially available PCR reagents in the unadded (control group, top) and added (experimental group, bottom) groups, where the symbols... The position is the length of the target segment.

[0025] Figure 8A shows the comparison results of PCR detection of SARS-CoV-2 samples with the addition of the sugar nanomaterials described in this invention (** represents p<0.01, *** represents p<0.001; n=3); Figure 8B shows the comparison results of envelope protein (E) in PCR detection of SARS-CoV-2 mutant samples with omicron mutations with the addition of the sugar nanomaterials described in this invention (* represents p<0.05; n=3); Figure 8C shows the comparison results of primer dimers in PCR detection of ALDH2(E487K) SNP mutant samples with the addition of the sugar nanomaterials described in this invention (* represents p<0.05; n=3).

[0026] Figure 9A shows the exponential growth curve (left) and melting curve analysis (right) of real-time PCR detection for the 2000 PFU virus concentration group; Figure 9B shows the exponential growth curve (left) and melting curve analysis (right) of real-time PCR detection for the 200 PFU virus concentration group; Figure 9C shows the Ct value analysis of real-time PCR detection for samples with different virus concentrations. Detailed Implementation

[0027] The following examples illustrate embodiments of the present invention. Those skilled in the art can easily understand the advantages and effects of the present invention from the disclosure disclosed herein. The present invention can also be implemented or applied through other different embodiments. Various details in this specification can also be modified and altered based on different viewpoints and applications, without departing from the scope disclosed in the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and are used in the context to describe the invention. The terminology used in this specification is for describing particular embodiments only and is not intended to limit the invention.

[0029] It should be noted that, as used herein, the singular terms “a,” “an,” and “the” include a plural of indicators unless explicitly limited to one. Unless the context clearly indicates otherwise, the term “or” is used interchangeably with the term “and / or.”

[0030] As used herein, the terms “about,” “approximately,” or “nearly” essentially mean that the stated value or range is within 5%, preferably within 3%, and more preferably within 1%. The numerical values ​​provided herein are approximate and are intended to be inferred even if the terms “about,” “approximately,” or “nearly” were not used.

[0031] As used herein, the term "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the term "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The term "substantially consisting of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially alter the essential characteristics of such embodiments.

[0032] In one embodiment, the present invention provides the use of a sugar nanomaterial for inhibiting DNA polymerase, wherein the sugar nanomaterial is generated by dry heating of sugars at 150°C to 300°C, wherein the sugar nanomaterial comprises graphene-like nanosheets and the sugars, wherein the graphene-like nanosheets comprise at least a portion of the carbonized products of the sugars, and the graphene-like nanosheets and the sugars are combined to form a cross-linked supramolecular structure.

[0033] In at least one embodiment, dry heating is performed at a temperature between 150°C and 300°C, for example, a lower limit of not less than 150°C or an upper limit of not more than 300°C. In some specific embodiments, the heating temperature is in the range of 180°C to 300°C, for example, approximately 190°C, approximately 200°C, approximately 210°C, approximately 220°C, approximately 230°C, approximately 240°C, approximately 250°C, approximately 260°C, approximately 270°C, approximately 280°C, and approximately 290°C.

[0034] In some embodiments, the sugar is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and their derivatives. In some embodiments, the sugar precursor is selected from the group consisting of hydroxyl and carboxyl groups. In some embodiments, the sugar is selected from the group consisting of tetracarbon sugar monomers, tetracarbon sugar repeating units, pentose sugar monomers, and pentose sugar repeating units.

[0035] In some specific embodiments, the sugar is selected from the group consisting of glucose, maltose, dextrin, sodium alginate, agarose, hydroxyethyl cellulose and xanthan gum.

[0036] In some specific embodiments, the surface of the cross-linked supramolecular structure has functional groups selected from the group consisting of hydroxyl, ester, phenol, carboxyl, and any combination thereof. The sugar nanomaterials of the present invention are obtained through the condensation of sugar ring opening and closing and the reaction regulation of benzene cyclization to obtain a complex cross-linked supramolecular structure with polycyclic aromatic phenols.

[0037] In some specific embodiments, the sugar nanomaterial inhibits the activity of the DNA polymerase by binding to it. Specifically, the sugar nanomaterial inhibits polymerase activity by binding to the active site of the DNA polymerase through its functional groups, and this inhibition is reversible.

[0038] In some specific embodiments, the sugar nanomaterial is used for a thermally initiated polymerase chain reaction. It binds to DNA polymerase to inhibit its activity, and releases the DNA polymerase by increasing the reaction temperature, thus restoring its activity. The inhibition of DNA polymerase activity by the sugar nanomaterial described in this invention is reversible; changes in temperature affect the binding or release of DNA polymerase by the sugar nanomaterial, therefore the inhibitory activity can be altered by controlling the temperature.

[0039] In another embodiment, the present invention provides a method for improving the precision of polymerase chain reactions by adding a sugar nanomaterial to the polymerase chain reaction. The sugar nanomaterial is generated by dry heating of sugars at 150°C to 300°C. The sugar nanomaterial comprises graphene-like nanosheets and the sugars, wherein the graphene-like nanosheets contain at least a portion of the carbonized products of the sugars, and the graphene-like nanosheets and the sugars are combined to form a cross-linked supramolecular structure. This precision includes both sensitivity and specificity.

[0040] In at least one embodiment, the sugar nanomaterial acquires thermally initiated properties by binding to DNA polymerase to inhibit its activity and releasing the DNA polymerase by increasing the reaction temperature to restore its activity.

[0041] In at least one embodiment, the concentration of the sugar nanomaterial is in the range of 1 μg / ml to 2500 μg / ml, for example, approximately 3 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml, 55 μg / ml, 60 μg / ml, 65 μg / ml, 70 μg / ml, 75 μg / ml, 80 μg / ml, 85 μg / ml, 90 μg / ml, 95 μg / ml, 100 μg / ml, 150 μg / ml, 200 μg / ml, 250 μg / ml, 300 μg / ml, 350 μg / ml, 400 μg / ml, 450 μg / ml. g / ml, 500μg / ml, 550μg / m, 600μg / ml, 650μg / ml, 700μg / ml, 750μg / ml, 800μg / ml, 8 50μg / ml, 900μg / m, 950μg / ml, 1000μg / ml, 1100μg / ml, 1200μg / ml, 1300μg / ml, 140 0μg / ml, 1500μg / ml, 1600μg / ml, 1700μg / ml, 1800μg / ml, 1900μg / ml, 2000μg / ml, 2 200μg / ml, 2250μg / ml, 2300μg / ml, 2350μg / ml, 2400μg / ml, 2450μg / ml and 2500μg / ml.

[0042] In some specific embodiments, the polymerase cascading reaction includes conventional polymerase cascading reaction, hot-start polymerase cascading reaction, capillary electrophoretic polymerase cascading reaction, instantaneous polymerase cascading reaction, multiplex polymerase cascading reaction, or allele-specific polymerase cascading reaction.

[0043] As used herein, the term "sugar nanomaterials" refers to carbonized products obtained by the dry heating method of sugars described in this invention. Under high temperature conditions, sugar precursors are decomposed, rearranged, and form carbon-based nanomaterials. In the bottom-up thermal synthesis method for preparing carbon quantum dots (CQDs) and carbonized polymer dots (CPDs), structural domains containing various functional groups are formed and embedded in the surface or structure of the material. These functional groups endow the material with specific chemical or physical properties.

[0044] As used herein, a “polymerase cascade reaction” must include at least template DNA, forward primers, reverse primers, deoxynucleoside triphosphates (dNTPs), DNA polymerase, and buffer solutions. As used herein, the term “hot-start polymerase cascade reaction” refers to the temporary inhibition of DNA polymerase activity at the beginning of a PCR reaction to prevent nonspecific amplification and primer dimer formation at low temperatures, until the PCR temperature rises to a certain level, at which point DNA polymerase activity is restored, thereby improving the accuracy of the amplification results. As used herein, the term “hot-start” refers more broadly to the process by which DNA polymerase becomes active through increased temperature.

[0045] As used herein, the term "carbohydrate" refers to aldehydes or ketones having at least two hydroxyl groups, their condensation polymers, or derivatives thereof, primarily composed of carbon, hydrogen, and oxygen, and includes, but is not limited to, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. As used herein, the term "carbohydrate derivative" refers to compounds derived from carbohydrates through chemical reactions or modifications. These derivatives typically retain the structural characteristics of the original carbohydrate molecule but undergo chemical changes at certain positions (e.g., substitution, oxidation, reduction, or combination with other groups), for example: sorbitol (derived from glucose reduction), gluconic acid (derived from glucose oxidation), or glucosamine (where the hydroxyl group at the C2 position of glucose is replaced by an amino group). As used herein, the term "carbohydrate precursor" refers to the carbohydrates used in the dry heating preparation of nanomaterials as described in this invention.

[0046] As used herein, the term "reversible" means that the binding of substances can be broken with changes in external conditions without permanently altering their structure or function. In other words, the binding between substances can be reversed. For example, the sugar nanomaterials of this invention bind to DNA polymerase, which can release DNA polymerase by increasing the temperature without changing the structure or activity of the DNA polymerase.

[0047] As used herein, the term "precision" refers to the ability to accurately amplify the target sequence fragment in each reaction while avoiding the amplification of non-target fragments or the generation of non-specific products. As used herein, the term "enhancing the precision of polymerase chain reaction" refers to improving the sensitivity and specificity of PCR reactions, increasing the amplification of the target fragment, and reducing the generation of non-specific products.

[0048] The present invention is further illustrated by the following embodiments, which are merely illustrative and not intended to limit the scope of the invention in any way. Materials used in the invention but not described herein are commercially available.

[0049] Example 1. Preparation method of sugar nanomaterials

[0050] This embodiment synthesizes graphene-like nanosheet-embedded sugar nanomaterials by heating a sugar precursor. Referring to Figure 1, the sugar precursor undergoes ring-opening hydration after dry heating. After ring-opening, the sugar precursor undergoes polymerization and crosslinking, and further condensation and aromatization, ultimately producing the sugar nanomaterials described in this invention.

[0051] [Amended according to Rule 26, 03.01.2025] In this embodiment, 50 mg of the carbohydrate precursor was placed in a 20 mL glass sample vial and dry-heated for 3 hours at 150°C, 200°C, 250°C, or 300°C in a laboratory-grade convection oven (DH 300, Dengyng, Taiwan, China). The obtained solid product was cooled and then dissolved in 5.0 mL of deionized water by ultrasonic vibration for 1 hour. Larger particles were removed by centrifugation at 500 g relative centrifugation force (RCF) for 30 minutes. The resulting carbohydrate nanomaterial dispersion was stored at 4°C for future use.

[0052] In this embodiment, glucose, maltose, dextrin, sodium alginate, agarose, hydroxyethyl cellulose, and xanthan gum were used as sugar precursors, and sugar nanomaterials were prepared at temperatures of 150℃, 200℃, 250℃, or 300℃. The yields after pyrolysis are shown in Table 1, and the yields of the soluble carbonized supernatant obtained after centrifugation purification are shown in Table 2.

[0053] Table 1. Pyrolysis yields of different sugars at different temperatures

[0054] Table 2. Yields of soluble carbonized products obtained from different sugars after treatment at different temperatures

[0055] Example 2. Inhibitory effect of DNA polymerase

[0056] In this embodiment, sugar nanomaterials prepared with different sugars and different heating temperatures were polymerized with DNA polymerase to detect the effect of sugar nanomaterials on the activity of DNA polymerase.

[0057] Materials and Methods

[0058] The method for preparing sugar nanomaterials in this embodiment is as described in Example 1.

[0059] [Amended according to Rule 26, 03.01.2025] Using 49 units of hairpin DNA as the acceptor, DNA polymerase uses this hairpin DNA as a template for polymerization. If the polymerization reaction proceeds normally, 64 units of hairpin DNA will be generated. Conversely, if the DNA polymerase activity is inhibited, 64 units of hairpin DNA cannot be generated. The DNA polymerase used in this experiment is Taq DNA polymerase (Bio-Helix, China Taiwan). The DNA polymerase activity was measured at 35°C for 80 minutes. Sugar nanomaterials prepared from glucose (dry heating temperatures of 150℃ and 200℃), maltose (dry heating temperatures of 150℃ and 200℃), dextrin (dry heating temperatures of 150℃ and 200℃), sodium alginate (dry heating temperatures of 150℃, 200℃, 250℃ and 300℃), agarose (dry heating temperatures of 150℃, 200℃ and 250℃), hydroxyethyl cellulose (dry heating temperatures of 150℃ and 200℃), and xanthan gum (dry heating temperatures of 150℃ and 200℃) were tested. After the reaction was completed, the amounts of the two types of hairpin DNA and their respective extension products were analyzed by capillary gel electrophoresis (CGE). The activity of DNA polymerase was quantified by calculating the peak area ratio of the 64-mer product to the 49-mer substrate.

[0060] Experimental results

[0061] Please refer to Figure 2. Sugar nanomaterials prepared from different sugar precursors at different heating temperatures all exhibit concentration-dependent inhibitory effects on DNA polymerase, with sodium alginate showing the best inhibitory effect when dry-heated at 250℃ and 300℃.

[0062] Example 3. Qualitative analysis of sugar nanomaterials

[0063] In this embodiment, sodium alginate was used as a sugar precursor. The effects of dry heating at temperatures of 150°C, 200°C, 250°C, and 300°C on the characteristics of sugar nanomaterials were analyzed to qualitatively characterize the sugar nanomaterials described in this invention.

[0064] Alginate is composed of D-mannuronic acid (M) units linked by β-(1→4) and L-glucuronic acid (G) units linked by α-(1→4). Sodium alginate is valued for its high biocompatibility, low cost, and the addition of divalent cations (e.g., Mg). 2+ and Ca 2+ Its moderate gel-forming ability has led to its widespread use in the food industry, pharmaceutical industry, and many biomedical applications, such as wound dressings, drug delivery, and immunotherapy.

[0065] Materials and Methods

[0066] The method for preparing sugar nanomaterials in this embodiment is as described in Example 1.

[0067] The particle size and morphology of sugar nanomaterials made from sodium alginate were analyzed using a transmission electron microscope (TEM) system operated at 200 kV by a Tecnai G2F20S-TWIN (Philips / FEI, Hillsboro, OG, USA).

[0068] The binding energy was corrected using the C1s peak at 284.6 eV as a standard. Fourier transform infrared spectroscopy (FT-IR, FT / IR-6100, JASCO, Easton, MD, USA) was used at 500 cm⁻¹. -1 Up to 4,000cm -1 Sixteen scans were performed in transmission mode to analyze functional groups that may already exist in sodium alginate nanomaterials.

[0069] Experimental results

[0070] Please refer to Figure 3A. The upper figure shows the carbonized products generated after dry heating of sodium alginate at 150°C, 200°C, 250°C, and 300°C. The lower figure shows the solution after dissolving it in deionized water and purifying it by centrifugation. Please refer to Figure 3B. Sodium alginate nanomaterials heated to 150°C contain gel-like cross-linked polymers formed by condensation reaction. In contrast, sodium alginate nanomaterials heated to 200°C to 300°C exhibit unique particles formed together with the polymer matrix, showing that the morphology of the sugar nanomaterials is highly correlated with the heating temperature. Please refer to Figure 3C. Sodium alginate nanomaterials heated to temperatures above 200°C exhibit a small polymer network and a clear graphitic carbon layer with a d-spacing value of 0.24 nm, showing (100) and (112) lattice planes and revealing that in addition to the cross-linked nanogel matrix, a structure similar to crystalline graphene is also formed.

[0071] Please refer to Figure 4. Fourier transform infrared spectroscopy analysis shows that when the synthesis temperature increased from 200℃ to 300℃, the CO extension peak was at 1035 / 1083 cm⁻¹. -1 The concentration of sodium alginate nanomaterials decreased significantly at 1330 cm⁻¹. Furthermore, the spectrum of sodium alginate nanomaterials at 1330 cm⁻¹ was significantly reduced. -1 There is a peak at a certain point, which is attributed to the bending of the -OH group of the phenolic group due to mild aromatization during carbonization. Therefore, the experimental results indicate that the sugar nanomaterials of the present invention are mildly carbonized, thereby retaining some functional groups and forming new functional groups, and show that the sugar nanomaterials of the present invention are rich in specific functional groups, such as hydroxyl, ester, phenol and carboxyl groups.

[0072] Example 4. Effects of polymerase chain reaction

[0073] In this embodiment, polymerase chain reaction (PCR) was performed using Taq DNA polymerase, and the sugar nanomaterials described in this invention, prepared at different temperatures, were added to the reaction. Subsequently, the effect of the sugar nanomaterials on PCR was visually evaluated by gel electrophoresis imaging.

[0074] 4.1 Sodium alginate nanomaterials

[0075] In this experiment, sodium alginate was dry-heated to form sodium alginate nanomaterials, which were then added to the PCR reaction to analyze their effects.

[0076] Materials and Methods

[0077] The method for preparing sugar nanomaterials in this embodiment is as described in Example 1.

[0078] PCR was performed using 30 ng of 406 bp human gDNA as a template. Sodium alginate nanomaterials, prepared by dry heating at 150℃, 200℃, 250℃, and 300℃, were added to the reaction at concentrations of 1, 10, and 100 μg / mL, respectively. Subsequent analysis was performed by colloidal electrophoresis. In the figures, M represents the DNA ladder band (DNA Ladder, Bio-Helix, Taiwan), C represents the control group without sodium alginate nanomaterials, and the unheated group received sodium alginate without dry heating.

[0079] [Amended according to Rule 26, 03.01.2025] The PCR reaction involves mixing different concentrations of the sugar nanomaterials described in this invention with 2 μL of 10X PCR buffer (Bio-Helix, Taiwan), 200 μM of dNTPs, 5 U of Taq DNA polymerase (Bio-Helix, Taiwan), 1000 copies of human genomic DNA template (Merck KGaA, Germany), and 500 nM of forward and reverse primers to a final volume of 20 μL. The PCR reaction is performed for 35 cycles, each cycle including denaturation at 95°C for 30 seconds, bonding at 54 or 64°C for 30 seconds, and extension at 72°C for 60 seconds. The amplified PCR products were separated by gel electrophoresis using 2% agar colloid and DNA ladder bands (DM115-0100, Bio-Helix, Taiwan, China). The products were then stained with HealthView nucleic acid staining agent (Genomics, Taiwan, China) and subsequently visualized under ultraviolet light.

[0080] Experimental results

[0081] Referring to Figure 5A, in the control group (C) without sodium alginate nanomaterials, it can be seen that in addition to generating the correct 406bp target DNA main product (target fragment), the PCR reaction also generates many non-specific byproducts. This is because the DNA polymerase has an excessively wide activity temperature range, causing it to react even at low temperatures, thus generating non-specific products. However, when sodium alginate nanomaterials are added to the PCR reaction, the expression of non-specific byproducts decreases. In particular, no non-specific byproducts are generated when sodium alginate nanomaterials at concentrations of 100 μg / mL generated at 200℃, 10 μg / mL generated at 250℃, and 10 μg / mL generated at 300℃ are added. Only the specific amplification of the 406bp target fragment is clearly observed, indicating that the specificity and sensitivity of DNA polymerase are greatly improved under these conditions. This specific amplification effect is similar to hot-start PCR using antibody-inhibited polymerase. In addition, sodium alginate nanomaterials generated at 250℃ and 300℃ have a strong binding ability with Taq DNA polymerase and have a better inhibitory effect, resulting in no PCR amplification at high concentrations (100μg / mL).

[0082] 4.2 Glucose Nanomaterials

[0083] In this experiment, glucose was dry-heated to form glucose nanomaterials, which were then added to the PCR reaction to analyze their effects.

[0084] Materials and Methods

[0085] The method for preparing sugar nanomaterials in this embodiment is as described in Example 1.

[0086] PCR was performed using 30 ng of 406 bp human gDNA as a template. Glucose nanomaterials, generated after dry heating at 150℃, 180℃, and 210℃, were added to the reaction at concentrations of 1.25, 2.5, and 5 mg / mL, respectively. Colloidal electrophoresis was then performed. In the figures, M represents the DNA ladder, C represents the control group (n=3) without glucose nanomaterials, and the unheated group received glucose without dry heating. The PCR reaction conditions were as described previously.

[0087] Experimental results

[0088] Please refer to Figure 5B. In the control group (C) without added glucose nanomaterials, the 406bp correctly labeled DNA main product (target fragment) was not clearly expressed, and many non-specific byproducts were generated. However, when 2.5 mg / mL of glucose nanomaterials generated at 180℃ and 2.5 mg / mL of glucose nanomaterials generated at 210℃ were added to the reaction, the non-specific byproducts disappeared, and only the obvious target fragment appeared, showing that the specificity of the PCR reaction was greatly improved.

[0089] Example 5. DNA polymerase inhibitory efficacy under different conditions

[0090] In this embodiment, different concentrations of the sugar nanomaterials described in this invention were reacted with Taq DNA polymerase, and different reaction temperatures were adjusted to confirm the inhibitory effect of the sugar nanomaterials described in this invention on DNA polymerase at different concentrations and temperatures.

[0091] Materials and Methods

[0092] The method for preparing sugar nanomaterials in this embodiment is as described in Example 1, wherein the sugar precursor used in this embodiment is sodium alginate, and the dry heating temperature is 250°C.

[0093] The DNA polymerase activity assay in this embodiment is as described in Example 2. The DNA polymerase was reacted for 80 minutes with 0 μg / mL, 2.5 μg / mL, 10 μg / mL or 40 μg / mL of the sugar nanomaterial described in this invention at 35°C, 45°C, 55°C, 65°C or 75°C, respectively. The group with 0 μg / mL of sugar nanomaterial added (without addition) was used as the control group.

[0094] Experimental results

[0095] Please refer to Figure 6. The group with 40 μg / mL sodium alginate nanomaterials significantly inhibited DNA polymerase activity at any temperature compared to the group without sodium alginate nanomaterials. Notably, at 35°C, the group with 10 μg / mL sodium alginate nanomaterials effectively inhibited Taq DNA polymerase activity (>99%), and this inhibitory effect was maintained at reaction temperatures of 45°C, 55°C, and 65°C. However, at a reaction temperature of 75°C, the DNA polymerase activity was significantly increased compared to the 65°C reaction temperature, recovering to approximately 80% of the level of the control group. This indicates that at a reaction temperature of 75°C, DNA polymerase is not inhibited by sodium alginate nanomaterials. Furthermore, the group with 2.5 μg / mL sodium alginate nanomaterials also showed a trend of increasing activity with increasing temperature, demonstrating that the sugar nanomaterials described in this invention can effectively inhibit DNA polymerase activity at low temperatures, but do not inhibit DNA polymerase activity at high temperatures.

[0096] PCR primarily relies on Taq DNA polymerase to catalyze the polymerization reaction at temperatures above 70°C. Therefore, the sugar nanomaterials described in this invention can effectively inhibit DNA polymerase, preventing unnecessary amplification during PCR sample preparation and amplification. Furthermore, experiments have shown a positive correlation between the dosage concentration of sodium alginate nanomaterials and their inhibitory effect on DNA polymerase activity. Therefore, the activity of DNA polymerase can be controlled by adjusting the concentration of sodium alginate nanomaterials.

[0097] Example 6. Efficacy of hot-start polymerase chain reaction

[0098] In this embodiment, the sugar nanomaterials described in this invention were added to a polymerase chain reaction to observe their effects on the specificity, sensitivity, and accuracy of the PCR reaction. The results were compared with commercially available hot-start polymerase chain reaction kits, and the effects of the sugar nanomaterials described in this invention on different types of DNA polymerases were further investigated.

[0099] The method for preparing the sugar nanomaterials in this embodiment is as described in Example 1, wherein the sugar precursor used in this embodiment is sodium alginate, and the dry heating temperature is 250°C. Unless otherwise mentioned, the PCR reaction conditions in this embodiment are as described in Example 4, and the reaction conditions for different PCR kits are performed according to the operating manual of each kit.

[0100] 6.1 DNA template concentration test

[0101] This experiment tested the effects of the sugar nanomaterials described in this invention and different template concentrations on PCR amplification.

[0102] Materials and Methods

[0103] Polymerase chain reaction (PCR) was performed using 500 bp human gDNA samples in lanes 1 (30), 2 (3), 3 (3), 4 (4), and 5 (0 ng). The experimental group received sodium alginate nanomaterials at a concentration of 10 μg / mL, while the control group did not receive sodium alginate nanomaterials. The reaction was then performed by colloidal electrophoresis. PCR reaction conditions are detailed in Example 4.

[0104] Experimental results

[0105] Please refer to Figure 7A. The amplification effect of the control group was not obvious under different template concentrations. In contrast, the groups with added sugar nanomaterials of the present invention showed significantly better amplification effects than the control group under the same conditions, indicating that the sugar nanomaterials of the present invention can effectively improve the amplification effect of PCR reaction.

[0106] 6.2 Concentration Test of Sugar Nanomaterials

[0107] This experiment tested the effect of adding different concentrations of the sugar nanomaterials described in this invention to the PCR reaction on the amplification.

[0108] Materials and Methods

[0109] 30 ng of 406 bp human gDNA was used as a template for polymerase chain reaction (PCR). Sodium alginate nanomaterials were added at concentrations of 0 (control group), 0.01, 0.1, 1, 10, and 100 μg / mL, respectively, followed by colloidal electrophoresis. The PCR reaction conditions are detailed in Example 4.

[0110] Experimental results

[0111] Please refer to Figure 7B. In the control group without the addition of the sugar nanomaterials described in this invention, the PCR reaction results produced many non-specific byproducts. In contrast, the group with the addition of the sugar nanomaterials described in this invention produced fewer non-specific byproducts. In particular, when 1 μg / mL of sodium alginate nanomaterials was added, the non-specific byproducts were significantly reduced. When 10 μg / mL of sodium alginate nanomaterials was added, the target fragment of the PCR reaction was significantly expressed, and all non-specific byproducts disappeared. This shows that the sugar nanomaterials described in this invention can effectively improve the specificity of the PCR reaction.

[0112] 6.3 DNA template length test

[0113] This experiment tests the effects of the sugar nanomaterials described in this invention and different template lengths on PCR amplification.

[0114] Materials and Methods

[0115] Human gDNA at lengths of 280 (lane 1), 320 (lane 2), 406 (lane 3), 653 (lane 4), and 941 (lane 5) bp was used as templates for polymerase chain reaction (PCR). 30 ng of DNA template was used. The experimental group received 10 μg / mL sodium alginate nanomaterial, while the control group received no sodium alginate nanomaterial. Subsequent PCR was performed using colloidal electrophoresis. PCR reaction conditions are detailed in Example 4.

[0116] Experimental results

[0117] Please refer to Figure 7C. The control group produced many non-specific bands after amplification, indicating that the DNA polymerase in the control group had poor specificity. The common reason is that the activity of DNA polymerase was not inhibited, resulting in non-specific polymerization at the beginning of the reaction. However, after adding the sugar nanomaterials described in this invention, no non-specific bands were produced, and the target length sequence could be significantly amplified. This shows that adding the sugar nanomaterials described in this invention can effectively improve the specificity and sensitivity of the PCR reaction and greatly increase the accuracy of the PCR reaction.

[0118] 6.4 Comparative Experiment of Commercially Available PCR Kits

[0119] This experiment compares the specificity and accuracy of PCR reactions with the added sugar nanomaterials of this invention with those of commercially available hot-start PCR kits.

[0120] Materials and Methods

[0121] Human gDNA at 320, 406, 653, and 941 bp was used as template for polymerase cascading reactions. 30 ng of DNA template was added to the glycosaminoglycan described in this invention (lane 1) for polymerase cascading reactions. Commercially available hot-start polymerase cascading reaction kits DreamTaq DNA polymerase (Thermo Scientific, USA) (lane 2), DreamTaq Hot Start DNA polymerase (Thermo Scientific, USA) (lane 3), and KAPA2G Fast HotStart PCR Kit (Roche, Switzerland) (lane 4) were used for PCR reactions under the same conditions, followed by colloidal electrophoresis. The PCR reaction conditions are detailed in Example 4.

[0122] Experimental results

[0123] Please refer to Figure 7D. Commercially available hot-start polymerase chain reaction kits (lanes 2, 3, and 4) all produce many non-specific amplification products. However, the group with added sugar nanomaterials of the present invention (lane 1) produces almost no non-specific bands, indicating that the sugar nanomaterials of the present invention can significantly improve the specificity of PCR reactions and are more effective than commercially available hot-start polymerase chain reaction kits.

[0124] 6.5 Commercially available PCR polymerase test

[0125] In this experiment, different commercially available PCR kits were supplemented with the sugar nanomaterials described in this invention, and PCR reactions were performed. Subsequently, colloidal electrophoresis was conducted to compare the effects of adding the sugar nanomaterials described in this invention on different DNA polymerases and amplification effects.

[0126] Materials and Methods

[0127] Polymerase cascading reactions were performed using 280, 320, and 406 bp human gDNA as templates. 30 ng of DNA template was used in each reaction. The control group did not contain the glyconanomaterial described in this invention, while the experimental groups contained 10 μg / mL of the glyconanomaterial described in this invention. The PCR detection kits were: Wild-type Taq (Wild Type Taq DNA polymerase, purchased from Ten Giga Bio, Taiwan), Bh-Taq (Taq DNA polymerase, purchased from Bio-Helix, Taiwan), BO-Taq (Gran Turismo PreMix, purchased from BiOptics, Taiwan), ExTaq (Ex Taq DNA Polymerase, purchased from TaKaRa, Japan), AzTaq (AZtaq DNA Polymerase, purchased from ArcticZymes, Norway), I-Taq (I-Taq DNA Polymerase, purchased from LiliF Diagnostics, South Korea), and I-StartTaq (I-StarTaq DNA Polymerase, purchased from LiliF Diagnostics, South Korea). Please refer to Example 4 for PCR reaction conditions. The reaction conditions for different PCR kits should be adjusted according to the operation manual of each kit.

[0128] Experimental results

[0129] Please refer to Figure 7E. In the control group without the addition of the sugar nanomaterials described in this invention, the commercially available PCR kits (BoTaq group, ExTaq group, AzTaq group, and I-Taq group) could not fully amplify the target lengths of 320 and 406 bp (control group, upper figure), and produced many non-specific bands. Although the I-StartTaq group could amplify the target sequence clearly, it also produced many non-specific bands. In contrast, after adding the sugar nanomaterials described in this invention to each PCR kit (experimental group, lower figure), the target sequences that could not be amplified before could be generated normally, and the amplification products were quite significant. Moreover, the non-specific amplification was significantly reduced, showing that the sugar nanomaterials described in this invention can be applied to different DNA polymerases, and after being added to different PCR kits, they can effectively increase the specificity and sensitivity of the PCR reaction and effectively improve the accuracy of the reaction.

[0130] Example 7. Applications and efficacy of PCR detection

[0131] This embodiment performs multiplex gene detection on SARS-CoV-2 virus and its variant (omicron) to explore the efficacy of the sugar nanomaterials described in this invention against background interference and virus detection. This embodiment further uses primer pairs for aldehyde dehydrogenase 2 (ALDH2) and its variant (E487K) to perform PCR reactions to explore the effect of the sugar nanomaterials described in this invention on primer-dimer, in order to analyze the efficacy of the sugar nanomaterials described in this invention for detecting single nucleotide polymorphisms (SNPs).

[0132] Materials and Methods

[0133] The method for preparing sugar nanomaterials in this embodiment is as described in Example 1, wherein the sugar precursor used in this embodiment is sodium alginate, and the dry heating temperature is 250°C.

[0134] Multiplex PCR detection of SARS-CoV-2 targets three viral genes: RNA-dependent RNA polymerase (RdRp), envelope protein (E), and nucleocapsid (N). Different target fragment sizes were selected for identification during capillary electrophoresis (CGE) separation: the target fragment for RdRp products was 100 bp, for envelope protein products it was 113 bp, and for nucleocapsid products it was 128 bp. Positive SARS-CoV-2 samples were obtained from Ten Giga Bio (Taiwan, China). Each reaction used 1000 copies of viral plasmid genes, 10 ng of human genomic DNA, 500 nM of primer mixture, and the sugar nanomaterials described in this invention for multiplex PCR, performing 45 cycles. Each cycle included denaturation at 95°C for 15 seconds, adhesion at 58°C for 20 seconds, and extension at 72°C for 30 seconds. The detection of SARS-CoV-2 variants employed a two-step multiplex PCR method, involving four reverse transcription PCR reactions. Each reaction contained 30 ng of human genomic DNA, 5 ng of total human RNA, and 10,000 replicates of synthetic RNA from the SARS-CoV-2 omicron variant (BiOptic, Taiwan). The primer pairs targeted the envelope protein of the omicron variant. The RT-PCR reagent (BiOptic, Taiwan) included 5X RT primers, a 2X RT premix, and an Rtase. Each 20 μL sample of the RT-PCR product was further used for multiplex PCR. The PCR reaction using the glyconanomaterials described in this invention consisted of 44 cycles, each cycle comprising denaturation at 95°C for 15 seconds, bonding at 65°C for 20 seconds, and extension at 72°C for 20 seconds. After the PCR reaction, the amplification products were mixed with 10 and 1000 kb DNA labels, and then analyzed by capillary electrophoresis using an S1 cartridge (Qsep100, BiOptic, Taiwan). The S / N signal of each target was quantified and compared using Q-Analyzer software (BiOptic, Taiwan). The experimental group consisted of the group with 10 μg / mL sodium alginate nanomaterials, while the control group consisted of the group without sodium alginate nanomaterials.

[0135] This embodiment further uses two sets of opposing primers to directly perform allele-specific direct-PCR on human saliva samples to detect single nucleotide polymorphisms (SNPs) of acetaldehyde dehydrogenase 2 (ALDH2) and observe the effect of adding the sugar nanomaterials described in this invention on primer dimers. The PCR reaction conditions and quantification methods are as described above.

[0136] Experimental results

[0137] Please refer to Figure 8A. Adding the sugar nanomaterials described in this invention significantly enhances the detection signals of RNA-dependent RNA polymerase gene (RdRp), envelope protein gene (E), and nucleocapsid protein gene (N) by at least 1.5 times, with the RdRp gene showing an enhancement of more than 2 times. This indicates that even under complex multiplex PCR detection conditions, the sugar nanomaterials described in this invention can effectively improve the specificity of the PCR reaction, thereby improving the overall detection accuracy. By simultaneously targeting multiple regions of the viral genome, it effectively improves the problem of false positives or false negatives. Furthermore, please refer to Figure 8B. In the detection of the SARS-CoV-2 variant (Omicron), adding the sugar nanomaterials described in this invention significantly enhances the PCR detection signal. The above results show that the sugar nanomaterials described in this invention can significantly improve the amplification effect even in the presence of human cDNA background interference during PCR reactions. Moreover, the sugar nanomaterials described in this invention can be applied to virus detection, further confirming that the sugar nanomaterials described in this invention can effectively improve the specificity of the PCR reaction and facilitate more accurate identification of viruses and their mutant strains.

[0138] Please refer to Figure 8C. In the detection of SNP variations of aldehyde dehydrogenase (ALDH), the groups with added sugar nanomaterials of the present invention showed significantly fewer primer dimer products, indicating that the sugar nanomaterials of the present invention can be applied to the detection of genetic diseases and can effectively improve the specificity of PCR reaction and improve the formation of primer dimers.

[0139] Example 8. Application and efficacy of qPCR detection

[0140] In this embodiment, enterovirus 71 (EV71) samples were tested by real-time PCR (qPCR) to analyze the efficacy of the sugar nanomaterials described in this invention for qPCR detection.

[0141] Materials and Methods

[0142] The method for preparing sugar nanomaterials in this embodiment is as described in Example 1, wherein the sugar precursor used in this embodiment is sodium alginate, and the dry heating temperature is 250°C.

[0143] Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using a viral nucleic acid extraction kit (purchased from BioHelix, Taiwan). The experimental procedures were performed according to the product's instruction manual. In this example, RNA was extracted from human cell lines infected with enterovirus 71 (EV71). The number of plaque-forming units (PFU) in the cell samples was estimated using a plaque formation assay. cDNA was then synthesized using an RT Premix (purchased from BiOptic, Taiwan). In this experiment, 20, 200, 2,000, or 20,000 PFU of RNA, 1X RT primers, and reverse transcriptase were reacted at 55°C for 15 minutes, followed by a reaction at 70°C for 15 minutes. The experimental group received 10 μg / mL of the glyconanomaterial described in this invention, while the control group did not.

[0144] Real-time PCR was performed using a qPCR MasterMix system (BioHelix, Taiwan). The experimental procedures were performed according to the product's instruction manual, and the final sample volume was adjusted to 20 μL. Thermal cycling was performed using an AriaMx real-time PCR system (Agilent, USA). The conditions were: pre-denaturation at 95°C for 5 minutes, followed by 40 amplification cycles. Each cycle consisted of 95°C for 30 seconds, 65°C for 30 seconds, 72°C for 1 minute, and a final extension at 72°C for 5 minutes.

[0145] Experimental results

[0146] Please refer to Figure 9A. In the group with a viral concentration of 2000 PFU, the exponential duplication curves of the samples show that the experimental group exhibits a more significant relative fluorescence signal compared to the control group, indicating that the addition of the sugar nanomaterials described in this invention can effectively enhance the amplification efficiency.

[0147] Referring to Figure 9B, in the 200 PFU viral concentration group, the exponential growth curves of the samples show that the experimental group exhibits a more significant relative fluorescence signal compared to the control group, indicating that the addition of the sugar nanomaterials described in this invention can effectively enhance amplification efficiency. Furthermore, melting curve assessments reveal that the control group (without added sugar nanomaterials) shows two distinct peaks, corresponding to the formation of the target amplification product and primer dimers, respectively. This indicates that the amplification curve of the control group contains non-specific signals from primer dimer formation. In contrast, the experimental group with added sugar nanomaterials effectively eliminates non-specific signals and prevents primer dimer formation, thereby ensuring more accurate Ct value readings.

[0148] As shown in Figure 9C, the cycle threshold (Ct value) of the control group sample with 2000 PFU was 28.24, while the cycle threshold (Ct value) of the experimental group sample with 2000 PFU was significantly reduced to 26.23, indicating that the sugar nanomaterials described in this invention can effectively improve the amplification efficiency of qPCR reactions. Furthermore, the Ct values ​​of the control group were similar in the 20 PFU and 200 PFU virus concentration groups. This is because the control group generated many non-specific products during amplification, affecting the accuracy of the Ct value. In contrast, the higher the virus concentration in the experimental group, the lower the Ct value, showing a clear linear relationship. This further demonstrates that adding the sugar nanomaterials described in this invention can improve the reliability of qPCR detection.

[0149] In summary, this invention discloses the preparation of sugar nanomaterials from sugar precursors using dry heating. These nanomaterials can be prepared from various sugars or their derivatives. The surface of these sugar nanomaterials possesses specific functional groups that effectively bind to DNA polymerase, thereby inhibiting its activity. This inhibition is reversible; as the temperature increases, the sugar nanomaterials release the bound DNA polymerase without affecting their structure or activity. Leveraging this characteristic, this invention further applies it to hot-start polymerase chain reactions. Experiments have demonstrated that the sugar nanomaterials of this invention effectively enhance the specificity and sensitivity of PCR reactions, while significantly reducing the generation of non-specific products and primer dimers, thus significantly improving the accuracy of PCR reactions. They can be applied in various fields such as molecular biology experiments, virus detection, and genetic testing. By enhancing reaction specificity and reducing non-specific amplification, the sugar nanomaterials of this invention enable more accurate quantification even at low concentrations. This enhanced specificity is crucial for the early detection of viral infections, allowing for more timely and effective disease progression management.

[0150] Furthermore, compared to commercially available hot-start polymerase chain reaction kits, the sugar nanomaterials described in this invention significantly enhance the specificity of PCR reactions, improve the amplification of target products, reduce the generation of byproducts, and inhibit primer dimer formation, thereby improving false positives or false negatives. They are also compatible with various DNA polymerase schemes and can be directly added to existing PCR detection kits to directly improve accuracy. On the other hand, the antibodies or aptamers used in traditional hot-start PCR are quite expensive; in contrast, the sugar nanomaterials described in this invention are very low-cost, easy to prepare, and also provide better PCR amplification.

[0151] The present invention has been disclosed above through the above embodiments, which are only some preferred embodiments of the present invention. However, they are not intended to limit the present invention. Any equivalent changes or modifications made by any person skilled in the art after understanding the foregoing technical features and embodiments of the present invention without departing from the spirit and scope of the present invention shall still fall within the scope of the present invention. The patent protection scope of the present invention shall be determined by the claims appended to this specification.

Claims

1. The use of a sugar nanomaterial for inhibiting DNA polymerase, characterized in that, The sugar nanomaterial is generated by dry heating of sugars at 150°C to 300°C. The sugar nanomaterial comprises graphene-like nanosheets and the sugars. The graphene-like nanosheets contain at least a portion of the carbonized products of the sugars, and the graphene-like nanosheets and the sugars are combined to form a cross-linked supramolecular structure.

2. The use as described in claim 1, characterized in that, The sugars are selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides and their derivatives.

3. The use as described in claim 1, characterized in that, The sugars are selected from the group consisting of glucose, maltose, dextrin, sodium alginate, agarose, hydroxyethyl cellulose and xanthan gum.

4. The use as described in claim 1, characterized in that, The surface of the cross-linked supramolecular structure has functional groups selected from the group consisting of hydroxyl, ester, phenol, carboxyl and any combination thereof.

5. The use as described in claim 1, characterized in that, This sugar nanomaterial inhibits the activity of the DNA polymerase by binding to it, and this inhibition is reversible.

6. The use as described in claim 5, characterized in that, This sugar nanomaterial is used for thermally initiated polymerase chain reactions. It binds to DNA polymerase to inhibit its activity and releases the DNA polymerase by increasing the reaction temperature, thus restoring its activity.

7. A method for improving the accuracy of polymerase chain reactions, characterized in that, It is achieved by adding a sugar nanomaterial to a polymerase chain reaction, wherein the sugar nanomaterial is generated by dry heating of sugars at 150°C to 300°C, wherein the sugar nanomaterial comprises graphene-like nanosheets and the sugars, wherein the graphene-like nanosheets comprise at least a portion of the carbonized products of the sugars, and the graphene-like nanosheets and the sugars are combined to form a cross-linked supramolecular structure.

8. The method as described in claim 7, characterized in that, This sugar nanomaterial inhibits the activity of DNA polymerase by binding to it, and then releases the DNA polymerase by increasing the reaction temperature, thus restoring its activity.

9. The use as described in claim 7, characterized in that, The concentration of the sugar nanomaterial ranges from 1 μg / ml to 2500 μg / ml.

10. The reagent as described in claim 7, characterized in that, This polymerase cascade reaction includes conventional polymerase cascade reaction, hot-start polymerase cascade reaction, capillary electrophoresis polymerase cascade reaction, instantaneous polymerase cascade reaction, multiplex polymerase cascade reaction, or allele-specific polymerase cascade reaction.

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