Aptamer for detection of aflatoxin m1 and process for preparation thereof

Abstract

The present invention relates to an aptamer for detection of Aflatoxin M1 mycotoxin where the aptamers comprise nucleotide sequence in any one or more of SEQ ID. No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, a homologous sequence or a functional fragment thereof. The present invention also provides a process for preparing said aptamers for detection of Aflatoxin M1 mycotoxin. The present invention also provides a kit comprising said aptamers for detection of Aflatoxin M1 mycotoxin and an electrochemical device comprising aptamers for detection of Aflatoxin M1 mycotoxin in food products.

Description

[0001] P W0100788

[0002] APTAMER FOR DETECTION OF AFLATOXIN Ml AND PROCESS FOR

[0003] PREPARATION THEREOF

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to aptamers for detection of mycotoxin. In particular, the invention relates to aptamer for detection of Aflatoxin Ml (AFM1) in various food samples. In addition, the invention relates to a process of preparing the aptamers.

[0006] BACKGROUND OF THE INVENTION

[0007] Ensuring food safety requires constant vigilance against harmful contaminants. Milk and milk derived food contamination poses a significant threat to human health, and one of the critical concerns is the presence of mycotoxins, such as aflatoxin Ml (AFM1), in various milk and dairy samples. Aflatoxin Ml (AFM1), a potent toxin produced by the fungus Aspergillus, poses a significant threat. This toxin contaminates milk, and milk derived food products. AFM1 originates when animals, particularly ruminants, consume Aflatoxin Bl (AFB 1). Accordingly, the contamination of food by aflatoxin, notably the potent carcinogen AFM1, presents a serious concern for both human health and food safety. Conventional detection methods often involve complex and time-consuming processes, hindering rapid and efficient screening of food commodities. Classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC), AFM1 is highly hazardous, exhibiting carcinogenic, teratogenic, and mutagenic properties in both humans and animals. Early and accurate detection of AFM1 contamination is paramount. Existing methods like PCR, ELISA, and HPLC serve this purpose, but they are often time-consuming and expensive.

[0008] For instance, US 11648259B2, reports the use of aptamers for mycotoxin detoxification in food and feed (Deoxynivalenol, Fumonisin, Ochratoxins, Zearalenone, and Aflatoxin Bl). However, this document does not discuss about the AFM1.

[0009] Another non-patent document Xunjiao Wei et al., (J. Agric. Food Chem. 2023); the development of aptamer against AFM1 by using Graphene oxide-SELEX (GO-SELEX). Seven rounds of repeated screening were performed to check the affinity and specificity and does not mention any negative SELEX which is necessary to avoid the cross -reactivity of aptamers to the other mycotoxins. P W0100788

[0010] In another non-patent document, Seyedeh Fatemeh Ahmadi et al., (Food Chemistry, Volume 373, Part A, 30 March 2022), the invention uses the commercially available thiol-modified AFM1 aptamer which was obtained from Bioneer (Daejeon, Korea) with the following sequence: 5'-SH-ACTGCTAGA GATTTTCCACAT-3'. However, the reported method is complex and time-consuming.

[0011] Another non-patent document Yi Zhang et al., (Microchemical Journal Volume 166, July 2021) discloses an invention which uses a commercially procured amino -modified ssDNA- aptamer5'-NH2-(CH2)6-ACTGCTAGAGATTTTCCACAT-3' (HPLC purified) was purchased from Sangon (Shanghai, China).

[0012] To overcome the shortcomings in existing technologies, there is a need to develop an improved aptamer-based technology for the detection of AFM1, which can provide accurate and sensitive results in a much faster and easier manner. Briefly, aptamers are single-stranded DNA molecules that bind specifically to target molecules like AFM1. This targeted approach offers a promising solution. Hence, there is an unmet need in the art to develop aptamer-based technology for the detection of AFM1 and to develop a robust, cost-effective method for AFM1 detection in food products.

[0013] OBJECTIVES OF THE INVENTION

[0014] The main objective of the present invention is to provide aptamers for detection of Aflatoxin Ml (AFM1) mycotoxin.

[0015] Another objective of the present invention is to provide a process for preparing the aptamers for detection of Aflatoxin Ml mycotoxin.

[0016] Yet another objective of the present invention is to provide a novel strategy having the potential to significantly improve food safety by enabling rapid and precise identification of AFM1 contamination.

[0017] SUMMARY OF THE INVENTION

[0018] Accordingly, in an aspect, the present invention relates to an aptamer having specific binding affinity to aflatoxin Ml(AFMl), wherein the aptamer comprises a nucleotide sequence having P W0100788

[0019] SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence having 80%-99% sequence identity or a functional fragment thereof.

[0020] In an embodiment, the present disclosure provides an aptamer having specific binding affinity to aflatoxin Ml(AFMl), wherein the aptamer comprises a nucleotide sequence having SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence or a functional fragment thereof. In an embodiment, the aptamer may be a single- stranded DNA (ssDNA) or a single-stranded RNA (ssRNA).

[0021] In an aspect, the present invention provides a process for the preparation of an aptamer having specific binding affinity to aflatoxin Ml(AFMl), wherein the process comprises the steps of:

[0022] (a) adding Aflatoxin Ml mycotoxin stock solution to an immune affinity column (IAC);

[0023] (b) adding an ssDNA library solution to said immune affinity column (IAC) and incubating for a specified period under specified conditions;

[0024] (c) eluting the IAC with nuclease free water or a suitable buffer to obtain the bound oligonucleotides:

[0025] (d) amplifying the eluent of step (c) by polymerase chain reaction (PCR) using suitable forward and reverse primers;

[0026] (e) subjecting the amplicon of step (d) to several iterations of positive SELEX in the presence of Aflatoxin Ml mycotoxin;

[0027] (f) subjecting the amplicon of step (e) to a number of iterations of negative SELEX against mycotoxins other than aflatoxin Ml (AFM1);

[0028] (g) digesting the dsDNA amplicon of steps (e) & (f) using suitable exonuclease at specified conditions;

[0029] (h) cloning and transforming the Aptamers to obtain the ssDNA Aptamers having Seq ID NO: 1-16.

[0030] The process may further comprise the steps as follows:

[0031] (i) (i) confirmation of the aptamer obtained in step (h) by PCR and sequencing; and

[0032] (j) (j) testing the developed aptamer obtained in step (i) against AFM1 toxin conjugate through aptamer-based ELISA (Enzyme Linked Immuno Sorbent Assay).

[0033] The present invention further envisages a composition comprising a plurality of aptamers specific to aflatoxin Ml, wherein said aptamers comprise nucleic acid sequences of SEQ ID. No. 1,2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16, a homologous sequence or a functional fragment thereof.

[0034] The invention further discloses a device for detecting AFM1 in a sample, comprising one or P W0100788 more aptamers of having nucleotide sequence of SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence or a functional fragment thereof, wherein the device may be a lateral flow device or lateral flow test strip or dip stick. In an aspect, said device may be an electrochemical sensor device comprising a redox labelled aptamer immobilized on the surface of the electrode, wherein the detection of AFM1 in the sample to be tested is achieved by measuring the electrochemical signal generated due to AFM1 -aptamer binding.

[0035] Further, the disclosure provides a kit for the detection of aflatoxin Ml(AFMl) in a sample, wherein the kit comprises one or more aptamers of nucleotide sequence having SEQ ID. No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence or a functional fragment thereof, along with an aptamer-functional substance complex, buffer solution, binding buffer, chromogenic dyes and instruction manual.

[0036] The present invention encompasses a method for the detection of aflatoxin Ml(AFMl) in a sample, the method comprising the steps of: providing a sample suspected of containing Aflatoxin Ml; conducting a detection assay utilizing single- stranded DNA (ssDNA) aptamers specific to Aflatoxin Ml as claimed in claim 1, followed by determining the presence or absence of Aflatoxin Ml in said sample based on the binding affinity of said aptamers.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG.l illustrates the final round of SEEEX PCR product resolved on 2% Agarose gel electrophoresis

[0039] FIG. 2 shows the confirmation of aptamers using vector specific primers resolved on Agarose gel electrophoresis (2%).

[0040] FIG.3 shows the amplified PCR product using aptamer specific primer resolved on agarose gel electrophoresis (2%).

[0041] FIG.4 illustrates the screening of novel ssDNA aptamers for their binding ability against AFM I .

[0042] FIG.5 shows quantitative determination of different concentration of aptamers against aflatoxin Ml by EEISA.

[0043] FIG.6 displays the quantitative ability of aptamer to bind to the different concentration of AFMI.

[0044] FIG.7A and 7B shows the secondary structure of Aflatoxin Ml aptamers from 1 to 16.

[0045] FIG.8 A to D shows nonlinear regression plot to calculate the binding affinity of the ligand with the aptamer. P W0100788

[0046] FIG.9A shows nyquist plot of EIS measurements with increasing concentrations of AFM1, plotted for detection of AFM1 and 9(B) shows the charge-transfer resistance (Ret) fitted against the concentrations of AFM1 to plot a linear graph for FOD calculation in buffer.

[0047] DETAILED DESCRIPTION OF THE INVENTION

[0048] The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

[0049] Aptamers are single- stranded DNA (ssDNA) or RNA (ss RNA) molecules that bind specifically to target molecules. They exhibit a range of affinities, with variable levels of off- target binding and are sometimes classified as chemical antibodies. This targeted approach offers a promising solution. By leveraging aptamers, the present invention has targeted to develop a robust, cost-effective method for AFM1 detection in food systems. This strategy has the potential to significantly improve food safety by enabling rapid and precise identification of AFM1 contamination.

[0050] In an aspect, the present invention relates to aptamer having specific binding affinity to aflatoxin Ml(AFMl), wherein the aptamer comprises a nucleotide sequence having SEQ ID. No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence or a functional fragment thereof. The homologous sequences may be sequences that share 80% to 99%, preferably 85% to 99%, preferably 90% to 99%, more preferably 95% to 99% sequence identity with SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16. The homologous sequence may be sequences that share 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0051] In yet another embodiment, the present invention provides aptamers having SEQ ID NO. 1-16, which are engineered or modified to improve specificity and sensitivity.

[0052] In an aspect, the present invention discloses ssDNA aptamers for AFM1 detection in food samples. The food samples may include any type of food such as meat, vegetables, fruits, dairy, bread and so on. The sample may be preferably dairy including milk or other dairy products.

[0053] In another aspect, the present invention discloses a process for the preparation of an aptamer having specific binding affinity to aflatoxin Ml(AFMl). The invention discloses a process for the preparation of an aptamer having nucleotide sequence having SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence or a functional fragment thereof.

[0054] In an embodiment, the present invention relates to process of preparing an aptamer having specific binding affinity to aflatoxin Ml(AFMl) comprising the steps of: P W0100788

[0055] (a) adding Aflatoxin Ml mycotoxin stock solution to an immune affinity column (IAC);

[0056] (b) adding an ssDNA library solution to said immune affinity column (IAC) and incubating for a specified period under specified conditions;

[0057] (c) eluting the IAC with nuclease free water or a suitable buffer to obtain the bound oligonucleotides:

[0058] (d) amplifying the eluent of step (c) by polymerase chain reaction (PCR) using suitable forward and reverse primers;

[0059] (e) subjecting the amplicon of step (d) to several iterations of positive SELEX in the presence of Aflatoxin Ml mycotoxin;

[0060] (f) subjecting the amplicon of step (e) to a number of iterations of negative SELEX against mycotoxins other than aflatoxin Ml (AFM1);

[0061] (g) digesting the dsDNA amplicon of steps (e) & (f) using suitable exonuclease at specified conditions;

[0062] (h) cloning and transforming the Aptamers to obtain the ssDNA Aptamers having Seq ID NO: 1-16.

[0063] The process may further comprise the steps as follows:

[0064] (i) confirmation of the aptamer obtained in step (h) by qPCR and sequencing; and

[0065] (j) testing the developed aptamer obtained in step (i) against AFM1 toxin conjugate through aptamer-based ELISA (Enzyme Linked Immuno Sorbent Assay).

[0066] In an embodiment, process of preparing an aptamer having specific binding affinity to aflatoxin Ml(AFMl) comprises the steps of

[0067] (a) adding 15-25pL Aflatoxin Ml mycotoxin stock solution (1-100 ng / mL) to an immune affinity column (IAC);

[0068] (b) adding 70-120pL ssDNA library solution (10-100 ng / mL) to said immune affinity column (IAC) and incubating for a period of 30 minutes to 1.5 hours at a temperature of 23-25 °C;

[0069] (c) eluting the immune affinity column (IAC) with nuclease free water to obtain the bound oligonucleotides:

[0070] (d) amplifying the eluent of step (c) by polymerase chain reaction (PCR) having non-biotin labelled forward primer (5’- ATGCGGATCCCGCGC3’), and biotinylated reverse primer (5’ Bio-GCGCAAGCTTCGCGC-3’);

[0071] (e) subjecting the amplicon of step (d) to seven-eight iterations of positive SELEX in the presence of Aflatoxin Ml mycotoxin; P W0100788

[0072] (f) subjecting the amplicon of step (e) to three-five iterations of negative SELEX against the zearalenone (ZEA), citrinin (CIT), and aflatoxin Bl (AFB1) mycotoxins;

[0073] (g) digesting the dsDNA amplicon of steps (e) & (f) with 10-100 U / mL of X-exonuclease enzyme at 25-40°C for 3-5 h;

[0074] (h) cloning and transforming the Aptamers to obtain the ssDNA Aptamers having Seq ID NO: 1-16;

[0075] (i) confirmation of the aptamer obtained in step (h) by PCR and sequencing; and

[0076] (j) testing the developed aptamer obtained in step (i) against AFM1 toxin conjugate through aptamer based Enzyme Linked Immuno Sorbent Assay (ELISA).

[0077] In an embodiment, Aflatoxin Ml mycotoxin stock solution added to an immune affinity column (IAC) in step (a) may be 15-25pL, preferably 20pL. The concentration of Aflatoxin Ml mycotoxin stock solution in step a) may be 1-100 ng / ml. In further embodiments it may be 20-100 ng / ml, 40-100 ng / ml, 50-100 ng / ml, 70-100 ng / ml or 80-100 ng / ml.

[0078] In step b), ssDNA library solution may be added to the immune affinity column (IAC) in an amount of 70-120pL, preferably 100 pL. The stock ssDNA library solution may have a concentration of 10-100 ng / mL. In further embodiments it may be 20-100 ng / mL, 40- lOOng / mL, 60-100ng / mL or 80-100ng / mL. The incubation in step b) may be performed for Ihr.

[0079] The digestion in steps (g) may be carried out at 25-40°C, preferably 37°C. The concentration of k-cxon uc lease enzyme may be 10-100 U / mL. In further embodiments it may be 20-100 U / mL, 40-100 U / mL, 60-100 U / mL or 80-100 U / mL.

[0080] In an embodiment, the aptamers are prepared using SELEX (Systematic Evolution of Ligands by Exponential enrichment) method. SELEX procedure is used to find the target aptamer against AFM1, the developed aptamer is evaluated for its specificity and reactivity towards the AFM1 by involving various rounds of negative SELEX against other mycotoxins. The target AFM1 SELEX product is cloned, sequenced, and further confirmed by ELISA. A total of sixteen aptamers were developed and showed more specificity and reactivity towards AFM1. In another embodiment, the developed ssDNA aptamer sequences are efficient to detect the AFM1 at nano-gram level using aptamer-based ELISA which is a semi-quantitative. The sensitivity can be improved using engineered aptamers, this would help in picogram level with easy electrochemical process. The sensitivity of developed aptamer towards AFM1 is evaluated by aptamer-based ELISA and showed high sensitivity ranged 10 to 50 ng. In another embodiment, the sensitivity of the developed aptamer may be about 50 ng. P W0100788

[0081] In yet another embodiment, the present invention provides a composition comprising a plurality of single- stranded DNA (ssDNA) aptamers specific to Aflatoxin, wherein said aptamers have nucleotide sequences of SEQ ID. No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 and isolated through SELEX and are capable of binding with high affinity and specificity to AFM1, thereby enabling the detection of Aflatoxin Ml in a sample.

[0082] In another embodiment, the present invention provides a kit for the detection of Aflatoxin Ml mycotoxin, comprising the ssDNA aptamers of the invention. The kit may comprise one or more aptamers having nucleotide sequence shown in any one of SEQ ID. No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, a homologous sequence or a functional fragment thereof. The kit for the detection of Aflatoxin Ml mycotoxin may further comprise an aptamer- functional substance complex, buffer solution, binding buffer, chromogenic dyes, and an instruction manual.

[0083] In another embodiment, the present invention relates to a device for detecting AFM1 in a sample. In an embodiment, the device is an electrochemical sensor-based device comprising the aptamers of the invention. The device may comprise an aptamer which is labelled (redox labelled aptamer), preferably with a dye on an internal site of the aptamer. Said labelled aptamer may be immobilized on an electrode surface. In the electrochemical sensor, the targetaptamer binding generates current signal change due to the alteration in distance between the redox tag and electrode surface and electron transfer efficiency of the redox tag to the electrode, enabling the detection of the target (AFM1).

[0084] In another embodiment, the present invention provides a method for monitoring and detecting Aflatoxin Ml contamination in milk and milk derived food products, comprising; obtaining a sample suspected of containing Aflatoxin Ml; and conducting a detection assay utilizing single- stranded DNA (ssDNA) aptamers specific to Aflatoxin Ml, followed by determining the presence or absence of Aflatoxin Ml in said sample based on the binding affinity of said aptamers.

[0085] These aptamers can be integrated into biosensors, known as Aptasensors, facilitating the development of accurate and straightforward analytical techniques. Aptasensors capitalize on the high sensitivity and selectivity of aptamers, enabling them to detect even trace amounts of AFM1 in milk and milk derived samples. This technology offers a significant advancement in AFM1 detection, as it combines the advantages of rapid results, ease of use, and adaptability to various detection platforms. In essence, aptamer-based AFMldetection holds immense potential in ensuring the safety and integrity of milk supplies, addressing a critical concern of public health and food security. P W0100788

[0086] It is worth noting that conventional detection methods often involve complex and timeconsuming processes, hindering rapid and efficient screening of milk and its derived products. The present invention developed aptamer-based technology for the detection of AFM1 with improve specificity and sensitivity.

[0087] In an embodiment, these aptamers are fabricated to lateral flow device or lateral flow test strip or dip stick. It uses simple, eco-friendly, system free, low cost. Further, the present invention aptamers can easily be used for the development of electrochemical aptasensor and lateral flow devices at low cost with user friendly features. Also, the LOD (limit of detection) of said aptamers were found as low as 5ng / mL using ELISA technique, which is considerably sensitive and effective than the reported in prior art. The LOD of said aptamers can go at much lower side if fluorescence technique is used for the detection.

[0088] In present invention, different ssDNA library used for aptamer development consisting of a 40 nucleotide (nt) randomized region with two flanking regions followed by SELEX procedure for screening of aptamer against the aflatoxin ML The SELEX procedure used for Aptamer Enrichment and Identification against AFM1 is simple, rapid and cost effective as compared to conventional fluorescence-labelled probe or group to detect AFM1 which are always more expensive and need system to record the data.

[0089] EXAMPLES

[0090] The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

[0091] Materials:

[0092] All the components and materials used in the present invention is commercially procured and easily available. The procurement details of the major components are provided below:

[0093] 1. 5 ’-biotinylated primer: Synthesized from Eurofins Pvt Ltd company, India.

[0094] 2. 5 '-phosphorylated reverse primer: Synthesized from Eurofins Pvt Ltd company, India

[0095] 3. Agarose gel: Invitrogen company, India

[0096] 4. Ampicillin: HiMedia company, India

[0097] 5. BSA: HiMedia company, India

[0098] 6. DNA loading dye: HiMedia company, India

[0099] 7. GoTaq® DNA Polymerase: Promega, USA P W0100788

[0100] 8. Forward primer 5’ATGCGGATCCCGCGC-3: Synthesized from Eurofins Pvt Ltd company, India

[0101] 9. Methanol, HPLC Grade: HI Media: catalogue no. AS061).

[0102] 10. Isopropanol (Hi Media)

[0103] 11. Zearalenone: Cayman Chemicals, USA

[0104] 12. Citrinin (CIT): Cayman Chemicals, USA

[0105] 13. Aflatoxin Bl (AFB1): Sigma Aldrich, USA

[0106] 14. LB medium: HiMedia company, India

[0107] 15. Lysozyme: HiMedia company, India

[0108] 16. MgCh: HiMedia company, India

[0109] 17. NaCl: HiMedia company, India

[0110] 18. pGEMT easy vector: Promega company, USA

[0111] 19. Phenol / chloroform / isoamyl alcohol (PCI): HiMedia company, India

[0112] 20. Immunoaffinity Coulmn VIC AM (Cat. No. G1007 Afla Ml)

[0113] 21. Reverse primer 5'GCGCAAGCTTCGCGC-3': Synthesized from Eurofins Pvt Ltd company, India

[0114] 22. E. coli DH5 alpha cells (cat No.JM109): Promega company, USA

[0115] 23. RNAse and DNAse: HiMedia company, India

[0116] 24. Skim milk powder: HiMedia company, India

[0117] 25. ssDNA library: Eurofins Pvt Ltd company, India

[0118] 26. Tris-HCl: Sigma Aldrich company, India

[0119] 27. Twin 20: HiMedia company, India

[0120] 28. Urea: HiMedia company, India

[0121] 29. k-cxonuclcasc enzyme: NEB, Germany

[0122] 30. Plasmid Extraction kit: Promega, USA

[0123] 31. PCR clean up and gel extraction (MACHEREY-NAGEL, Germany)

[0124] 32. Aflatoxin Ml: Sigma Aldrich, USA

[0125] 33. Microscale thermophoresis instrument

[0126] Example 1: ssDNA library amplification and ssDNA generation

[0127] The ssDNA library as a pool of random sequences or known as the randomized ssDNA library was purchased from Eurofins PVT. Ltd. India (Eurofins Genomics India PVT Ltd #540 / 1, Doddanakundi industrial area 2, Hoodi, Whitefield, Bengaluru 560048. India). This ssDNA library consisted of a 40 nucleotide (nt) randomized region with two flanking regions for use P W0100788 by the PCR primers (5’-ATGCGGATCCCGCGC-N40-GCGCAAGCTTCGCGC-3’) where N40 represents 40 nucleotides with equimolar incorporation of A, G, C, and T at each position.

[0128] All PCR reactions were carried out in a total volume of 100 pL with the forward primer (5'-

[0129] ATGCGGATCCCGCGC 3') and the reverse primer (5'-GCGCAAGCTTCGCGC-3').

[0130] (Eurofins Genomics India PVT Ltd #540 / 1, Doddanakundi industrial area 2, Hoodi, Whitefield,

[0131] Bengaluru 560048. India). The PCR mixture (10 pL) contained 5pL GoTaq polymerase (Promega, USA), 0.5 M of each of the forward and reverse primers, 100 ng of the template (ssDNA library). The mixture was thermally cycled 35 times at 95°C for 4 minutes, 94°C for

[0132] 30 seconds, and 60°C for 45 seconds, followed by a 1 -minute extension step at 72°C. The DNA library was amplified with the 5 ’-phosphorylated reverse primer (5'-P-

[0133] GCGCAAGCTTCGCGC 3'), for ssDNA generation. The reaction was then terminated by heating at 75 °C for 10 min.

[0134] The exonuclease product (Lambda Exonuclease (10 U / pL, Catalog number: EN0561 (Thermo Scientific, USA) was analyzed for quality by a Nanodrop spectrophotometer and 2% agarose gel electrophoresis.

[0135] Example 2: Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

[0136] The SELEX procedure for aflatoxin Ml (AFM1) was performed as per the method described by Setlem, K., Mondal, B., Ramlal, S., & Kingston, J. (2016). Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin BL Frontiers in microbiology, 7, 1909.

[0137] An immunoaffinity (IA) column (VIC AM, USA) was equilibrated three times with a 200pl binding buffer before use. The binding buffer comprised 10 mM Tris-HCl, pH 7.4, 1 M NaCl, and 10 mM MgCh. The AFM1 (Sigma Aldrich, India, catalogue no. A6428-250UG) stock solution was prepared in methanol (HPLC Grade, catalogue no. AS061). A 20 pL AFM1 stock solution was mixed with 80 pL binding buffer in an Eppendorf tube, resulting in a 100 pL solution of AFM1 solution. The AFM1 solution was then added to the equilibrated IA column and incubated for 30 minutes at 25°C. The obtained flow through was again passed through the column, and incubated for 30 minutes at 25°C and the same steps were repeated three times to increase the binding efficiency of the ssDNA to the AFM1 toxin in the column.

[0138] Concurrently, 100 pL of ssDNA library (10 pM) was kept in a water bath for 10 min at 95°C and immediately chilled on ice for 5 min. The 100 pl of the ssDNA library was then added to the IA column and incubated for 1 h at a temperature of 25°C. After incubation, 50 pL of binding buffer was added to the column to remove any unbound oligonucleotides. The bound P W0100788 oligonucleotides were then eluted by adding 100 pL of pre- warmed nuclease-free water. The eluted fraction was purified using an equal volume of isopropanol (Hi Media) and incubated at -20°C for 1 h. After incubation, the solution was centrifuged at 10,000 x g for 10-15 min, and the pellet obtained was air-dried and re-suspended in 20 pL of molecular biology-grade water. The obtained oligonucleotides were amplified using PCR for confirmation of the product, and followed by enrichment as discussed in the PCR amplification of the ssDNA library. Amplified products from each round were subjected to further selection rounds.

[0139] A total of 10 SELEX rounds / iterations were performed to increase the sensitivity and selectivity of the aptamers. A total of 7 positive SELEX rounds and 3 negative SELEX rounds were performed. The negative SELEX rounds were performed against zearalenone (ZEA), citrinin (CIT), and aflatoxin B l (AFB1) mycotoxins to negate the possibility of any crossreactivity due to their structural similarity with AFM1 in samples where high mycotoxin contamination is present. The protocol for negative SELEX was similar to the positive SELEX, except for the elution step. Briefly, after successful 5 positive SELEX rounds, negative SELEX was performed against zearalenone (ZEA) using zearalenone immunoaffinity column (VICM, Cat. No. G1012) and incubated for 1 h 25°C. A 50pL of binding buffer was added to the column to remove any unbound oligonucleotides. The bound oligonucleotides were then eluted by adding lOOpL of pre-warmed nuclease-free water. Herein, the unbounded ZEA fraction was collected and purified using an equal volume of isopropanol and incubated at -20°C for 1 h. After incubation, the solution was centrifuged at 10,000 x g for 10-15 min, and the pellet obtained was air-dried and re-suspended in 20 pL of molecular biology-grade water. The obtained oligonucleotides were amplified using PCR for confirmation of the product, and followed by enrichment as discussed in the PCR amplification of the ssDNA library. A similar procedure was followed with CIT, and AFB 1, and the same procedure was followed as discussed above.

[0140] Example 3: Polymerase Chain Reaction, and ssDNA generation

[0141] The eluent from the above 10thSELEX round was used as the PCR template. The PCR mixture (100 pL) contained 0.2 mM dNTPs, 0.5 pM of each of the forward and reverse primer, 100 ng template, and 2.5 U Taq DNA polymerase. The mixture was thermally cycled 35 times through 95°C for 4 min, 94°C for 30 sec, and 60°C for 45 sec and followed by a 1 min extension step at 72°C.

[0142] Subsequently, the double- stranded DNA amplicon (PCR product) was converted into single stranded DNA for the next round of SELEX. Hence, the PCR have been performed using P W0100788

[0143] 5 'phosphorylated reverse primer. The PCR conditions are the same as described in SELEX PCR. Purified dsDNA was incubated with 25 U X-cxonucleasc enzyme (Thermo fisher Scientific, USA) in a total reaction volume of 50 pL at 37°C for 3 h used for another PCR. The reaction was then terminated by adding 10 pl of DNA loading dye. The ssDNA products of the digested strand were analyzed by electrophoresis in a 2% agarose gel.

[0144] Example 4: Cloning and Sequencing of AFM1 aptamer in pGEM®-T Easy Vector

[0145] Cloning of the Aflatoxin Ml (AFM1) aptamer into the pGEM®-T Easy Vector system I (Promega Cat No. A1360). The procedure involves the confirmation of the aptamer band, ligation, transformation using E. coli DH5 alpha cells, confirmation of transformation through colony PCR, and subsequent plasmid isolation for further characterization and sequencing.

[0146] Confirmation of AFM1 Aptamer Band: A SELEX product was subjected to 2% Agarose gel electrophoresis, confirming the presence of a ~85 bp aptamer band (FIG.l). Ligation reactions were set up using 2X Rapid Ligation Buffer, pGEMT Vector, T4 DNA ligase, and insert DNA. Reactions were incubated overnight at 4°C.

[0147] Transformation: Competent E. coli DH5 alpha cells were transformed with the ligated product. The protocol involved thawing cells, heat shock, incubation, centrifugation, and spreading on LB plates containing IPTG, X-gal, and ampicillin.

[0148] Confirmation of transformation by colony PCR: Colonies were subjected to colony PCR using aptamer- specific primers. PCR parameters included initial denaturation, denaturation, annealing, elongation, and final elongation. The amplified products were analyzed on 2% Agarose gel electrophoresis.

[0149] Plasmid Isolation: Positive colonies were inoculated in LB broth containing ampicillin, followed by incubation and plasmid isolation using the MN Nucleospin Plasmid Mini kit. Isolated plasmids were confirmed on 2% Agarose gel and further purified using a PCR cleanup kit before being sent for sequencing.

[0150] Results: Colony PCR confirmed the successful transformation of the AFM1 aptamer into the pGEM®-T Easy Vector. Isolated plasmids were analyzed on 2% Agarose gel, showing the presence of the expected bands. Purified PCR products were sent for sequencing to verify the correctness of the cloned AFM1 aptamer.

[0151] The Aptamers sequences having SEQ ID. No. 1- 16 are shown below in Table 1:

[0152] Table 1. Aptamer sequence and Gibbs free energy change (AG) value of aptamer structure. [SEQ ID numbers of the Aptamers are given in brackets]. P W0100788

[0153] Note: For expression and amplification, the aptamer sequences are attached with forward primer (at 5 ’ end), and backward primer (at 3 ’ end).

[0154] Example 5: Development of aptamer-based ELISA for the detection of AFM1 aptamers

[0155] Coat plates with 100 pL per well of AFM1-BSA conjugate is provided followed by covering plates and incubate overnight (12-18 hours) at 2-8°C. Invert and tap on absorbent paper to remove excess liquid. Block plate is prepared in 100 pL per well with Blocking buffer (BSA 3%) for one hour at room temperature. Aspirate the blocking buffer then invert, and tap on absorbent paper to remove excess liquid. Aptamers were dissolved in CCB (Carbonate P W0100788 bicarbonate buffer). Aptamers were given a snap cold treatment before being added to the wells. Aptamers at the required concentration were first incubated at 95°C for 10 minutes before being refrigerated on ice for 5 minutes. Each aptamer was introduced to its corresponding well at a concentration of 50ul. Thereafter, incubated the same for two hours at 37 °C. Aspirate contents and wash wells three times with >100 pL of phosphate buffer saline tween 20 (PBST) per well. Following wash, invert and tap on absorbent paper to remove excess liquid. The working solution of streptavidin-HRP with blocking buffer (BSA 3%) is prepared by diluting in ratio of 1:5000. Add 100 pL of working streptavidin-HRP solution into each well, and incubate the same for one hour at 37°C. Aspirate the contents and wash wells three times with >100 pL of phosphate buffer saline pH 7.2 (PBS) per well. Following wash, invert and tap on absorbent paper to remove excess liquid. Add 100 pF of TMB substrate solution to each well. Incubate the plate for 10 minutes at room temperature or when the desired color intensity is reached. Add 100 pL of Stop solution (IN HCL) to each well and then, measured the absorbance at 450 nm within 30 minutes of adding the stop solution.

[0156] Example 6: Prediction of secondary structure of aptamers

[0157] The secondary structure of developed aptamers are designed with the help of UNAFold web server ("Unified Nucleic Acid Folding and hybridization package") (http: / / www.unafold.org / ) (Zuker, 2003) (FIG.7A and 7B). The server gives us an open access to prediction of secondary structure of DNA and RNA. The secondary structure will form by putting the FASTA sequence of the aptamer followed by formatting of the sequence. By keeping all other settings in default format, it gives a secondary structure and various other parameters like AG, different Circular structure Plots and predicted jpg file of aptamer. By using server, the aptamers for AMF1 are predicted and can be used for the further docking studies.

[0158] Example 7: Molecular Docking of AFM1 with designed aptamer

[0159] In this study, the binding interactions between Aflatoxin Ml and aptamers synthesized via Systematic Evolution of Ligands by Exponential enrichment (SELEX) was studied. Leveraging the High Ambiguity Driven Biomolecular Docking (HDOCK) server, docking analyses was conducted to unravel the intricate molecular interfaces governing the Aflatoxin Ml -aptamer complexes. The secondary structures of the aptamers were elucidated using the UNAFOLD web server, providing valuable insights into their 2D configurations. Prior to transitioning to 3D structural investigations using RNA Composer, a crucial pre-processing step involved the conversion of thymine (T) residues to uracil (U), aligning with the RNA P W0100788 molecule convention. This step ensures accurate representation and compatibility with RNA structural analyses. Subsequently, molecular docking results were scrutinized using BIOVIA and PYMOL, shedding light on the dynamic behavior and energetics of the Aflatoxin Ml- aptamer complexes. This comprehensive computational approach not only unveils the potential binding modes and affinities of Aflatoxin Ml with SELEX-derived aptamers but also provides a detailed structural understanding at both 2D and 3D levels. Insights gained from this study are anticipated to contribute significantly to the design and optimization of aptamer-based strategies for Aflatoxin Ml detection and mitigation in diverse food matrices, thereby addressing critical concerns related to food safety and public health.

[0160] Binding affinity of the docked models was assessed using knowledge-based scoring function, ITScorePP or ITScorePR. More negative scores indicate a stronger predicted interaction between the molecules. Based on the observation that protein-protein / RNA / DNA complexes in the Protein Data Bank (PDB) typically exhibit docking scores of -200 or lower, an empirical confidence score to assess the likelihood of two molecules binding was developed. This score is calculated as: Confidence score = 1.0 / [1.0 + exp (0.02 * (Docking Score + 150))].

[0161] This score provides a rough estimate of binding probability: Confidence score >0.7: High probability of binding. Confidence score between 0.5 and 0.7: Possible binding, but further investigation is recommended. Confidence score < 0.5: Unlikely to bind.

[0162] While ligand Root Mean Square Deviation (RMSD) is commonly used to assess docking model quality, it has limitations. It solely compares the final ligand pose in a model to the input or modeled structure, neglecting potential flexibility and conformational changes. Therefore, low RMSD doesn't guarantee the model accurately captures the true ligand-receptor interaction.

[0163] Example 8: Development of electrochemical-based sensor device

[0164] An electrochemical-based sensor device can be developed with aptamer with SEQ ID No. 10. The detailed workflow is as follows:

[0165] 1. The aptamer with SEQ ID No. 13 was labelled with a Cyanine5 (Cy5) dye group on a specific site of aptamer is immobilized on a gold electrode surface through the goldsulfur chemistry.

[0166] 2. In the absence of target AFM1, the Cy5 tag on the specific site shows slow electron transfer (eT) efficiency due to the possible interaction with aptamer bases or a relatively large distance from the electrode surface.

[0167] 3. AFB1 binding induces conformation change of aptamer, causing changes in the local environment of MB, interactions between Cy5 and aptamer bases, or distance of Cy5 P W0100788 to electrode, and brings Cy5 closer to the electrode surface, with enhancing electron transfer (eT) efficiency, so signal-on responses are generated.

[0168] 4. Screening a series of sites of the aptamer sequence helps to find such a proper position for MB labelling.

[0169] 5. Detection of target AFM1 is achieved by measuring the current change of Cy5.

[0170] 6. In the electrochemical sensor, the target-aptamer binding generates current signal change due to the alteration in distance between the redox tag and electrode surface and electron transfer efficiency of the redox tag to the electrode (Wang C, Zhao Q. Areagent less electrochemical sensor for aflatoxin Bl with sensitive signal -on responses using aptamer with methylene blue label at specific internal thymine.

[0171] Results

[0172] Cloning of AFM 1 Aptamer in pGEM®-T Easy Vector: For Cloning, SELEX product was taken and confirmed on 2% Agarose gel electrophoresis.

[0173] Ligation Aflatoxin Ml Aptamer Using the pGEM®-T Easy Vector: Ligation of AFM1 Aptamer (7 Ibp) in pGEM Easy Vector by using Promega Kit.

[0174] Transformation of ligated product in E.coli DH5 a high-efficiency competent cells: Transformation was performed and transformed cells were observed on AMP- LB Agar Plate Colony PCR performed for the confirmation using an aptamer- specific primers.

[0175] Plasmid Isolation: Positive colonies were grown in LB media for Plasmid Isolation and Plasmids were extracted by using Promega, USA Plasmid kit protocol. The extracted DNA were run on Agarose gel.

[0176] Plasmid: Aptamer Specific PCR: Isolated Plasmid were confirmed by Aptamer specific PCR and then run on Agarose gel electrophoresis

[0177] After confirmation on Agarose gel electrophoresis, isolated plasmid were sent for sequencing.

[0178] ELISA

[0179] The 16 novel aptamers were developed through SELEX and each aptamer is tested against AFM1 toxin conjugate through aptamer-based ELISA (FIG.4). The validation of these aptamers was done by absorbance at 450nm. As per the designed protocol out of 16 aptamers AFMl_APT10, AFM1_APT12, and AFM1_APT13 were selected which are showing maximum absorbance and verifies in triplicate.

[0180] The three aptamers showing maximum absorbance were again screened in triplicate, giving confirmation of the maximum binding aptamer. AFM1_APT13 showing maximum absorbance and thus selected for the further validation. P W0100788

[0181] After selecting AFM1_APT13 as the best aptamer, it was further used to detect the change in absorbance with respect to increasing aptamer concentration (FIG.5). The microtiter plates were coated with lOOpL of AFM1 conjugate (lOOng) with carbonate -bicarbonate buffer as blank, and incubated at 4°C overnight. Next day, lOOpL of 3% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) (w / v) was used to block the unbound sites, which was then incubated at room temperature for 1 hour. To this solution in each well, biotinylated aptamers in increasing volumes of selected ssDNA aptamer (1, 5, 10, 25, 50, 75, lOOng / well) were added sequentially for screening their ability to detect AFM1 and the plates were incubated for 2hrs at 37°C. Later, plate washed with Phosphate Buffered Saline with Tween was applied to the plates three times. Streptavidin-HRP conjugate (1:5000) was added, and the mixture was then incubated at 37°C for one additional hour. The plates were washed with PBS and lOOpL of TMB solution was added as a chromogenic substrate and blue coloration was developed on incubating for 15-30min at room temperature and to stop the reaction plate was treated with lOOpL IN HCL for which absorbance can be recorded at 450nm. The absorbance readings are directly proportional to the concentration of toxin present in the solution. The values were read at 450 nm after development.

[0182] Furthermore, for the detection of minimum concentration of toxin, aptamer-based ELISA was carried out with different toxin concentration (FIG.6). The microplate was coated with AMF1 conjugate with (1, 5, 10, 25, 50, 75, lOOng / well) and carbonate -bicarbonate buffer as blank, and incubated at 4°C overnight. Next day, lOOpL of 3% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) (w / v) was used to block the unbound sites, which was then incubated at room temperature for 1 hour. To this solution in each well, biotinylated aptamers lOOng / well were added sequentially for screening their ability to detect AFM1 and the plates were incubated for 2hrs at 37°C. Later, plate washed with Phosphate Buffered Saline with Tween was applied to the plates three times. Streptavidin-HRP conjugate (1:5000) was added, and the mixture was then incubated at 37 °C for one additional hour. The plates were washed with PBS and lOOpL of TMB solution was added as a chromogenic substrate and blue coloration was developed on incubating for 15-30min at room temperature and to stop the reaction plate was treated with lOOpL IN HCL for which absorbance can be recorded at 450nm. The absorbance readings are directly proportional to the concentration of toxin present in the solution. The values were read at 450 nm after development.

[0183] Aptamer’s secondary structure prediction using UNAFOLD web server. P W0100788

[0184] The secondary structure and Gibbs free energies (AG) of novel aptamer were analyzed using UNAFOLD online web server under physiological conditions (37°C, ImM [Na+], and 3 mM [Mg2+]). The chosen aptamer, designated as APT10, exhibited a remarkably low Gibbs free energy of -15.39 kcal / mol, indicative of its strong binding affinity towards AFM1 conjugate. This finding underscores the potential of APT 10 as a promising candidate for applications requiring high-affinity molecular recognition, such as in biosensing or targeted drug delivery systems.

[0185] Molecular Docking analysis of aptamer with the AFM1 toxin

[0186] The wealth of data generated below allowed for a comprehensive analysis of the binding effectiveness of various aptamers towards Aflatoxin Ml. Remarkable binding efficacy was exhibited by aptamer 13, signifying their potential as robust candidates for Aflatoxin Ml recognition. These results underscore the utility of computational approaches in elucidating the molecular nuances of aptamer- Aflatoxin Ml interactions, providing valuable insights that could pave the way for the development of highly effective diagnostic and therapeutic strategies. The specificity and high binding affinity demonstrated by aptamers 13 offer promising avenues for further exploration and application in addressing Aflatoxin Ml -related challenges in diverse fields, ranging from food safety to healthcare.

[0187] Example 9: Microscale Thermophoresis (MST) for Aptamer-AFMl Binding Affinity and Selectivity

[0188] The binding affinity of the aptamer toward Aflatoxin Ml (AFM1) was determined using microscale thermophoresis (MST), following established protocols with minor modifications (Wienken et al, .2010). Briefly, a 5' Cy5-labeled aptamer was prepared at a final concentration of 10 nM in MST buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCb, 0.05% Tween-20, pH 7.4). Serial 1 :2 dilutions of AFM1 were prepared to generate a range of concentrations covering at least two orders of magnitude around the expected dissociation constant (Kd). Each dilution (10 pL) was mixed with an equal volume of the labelled aptamer solution, incubated at room temperature for 20 minutes to allow equilibrium binding, and then loaded into twelve standard MST capillaries (NanoTemper Technologies).

[0189] The normalized fluorescence (Fnorm) of the aptamer-AFMl complex was measured for each titration point. The fraction of bound aptamer was calculated by normalizing the fluorescence signal of the complex against the unbound aptamer control. The binding curve was generated by plotting Fnorm against the logarithm of AFM1 concentration, and the dissociation constant P W0100788 was determined by fitting the data to a nonlinear regression model. To assess the selectivity of the aptamer, parallel MST experiments were conducted under identical conditions using homologous mycotoxins, including zearalenone (ZEA) and aflatoxin Bl (AFB), at concentrations equivalent to those used for AFM1. The resulting binding curves were analyzed and compared to evaluate the specificity of the aptamer for AFM1 over structurally related toxins.

[0190] Example 10: Fabrication of the Impedimetric Aptasensor for Detection of AFM1. Electrochemical impedance spectroscopy (EIS) was performed using a potentiostat three electrode system to develop AFM1 aptasensor following the methodologies described by Zhang et al. (2018) with certain modifications. The 5 mM K Fe (CN)e and 25 mM KNO3 electrolytic solution was applied as redox reporters to measure the electrochemical signal at the electrode surface. For EIS, each measurement was swept from 10 kHz to 0.1 Hz at a bias potential of 0.3 V with an alternating potential of 10 mV. The parameters in the equivalent circuit such as electrolyte resistance (Re), charge-transfer resistance (Ret), and constant phase element were determined from the model circuit using the software PSTrace 5.9. The optimum aptamer coating concentration, coating time, and the time required for aptamer-target binding were analyzed by recording the impedance signal. The specificity of the electrochemical AFM1 sensor was analyzed in a buffer spiked with AFM1 in varying concentrations and was used to estimate the limit of detection (LOD) for the aptasensor. From the Nyquist plot, the Ret of each sample was calculated by using the equation [Ret = (R - R0) / R0], where R is the impedance at each concentration of AFM1, while R0 is the impedance of the blank. The Ret values for each concentration of AFM1 were fitted into a linear regression plot to calculate the LOD using the equation [LOD = (3 x SD) / slope of blank].

[0191] Results

[0192] The specificity and binding affinity of Aptamer 13 (AFM1 APT 13) with AFM1 were analyzed using MST, which measures the molecular movement of the aptamer-target complex through a temperature gradient. A concentration-dependent change in fluorescence signal may be attributed to the alteration in one or more thermophoretic properties like charge, size, and hydration shell upon binding with AFM1. FIG.8 (A) shows the binding affinity by calculating signal to noise ratio of bound and unbound fractions of target and ligand. The binding of Aptamer 13 was determined at increasing concentrations of AFM1 (FIG.8B), AFB (FIG.8C), ZEA (FIG. 8D) by plotting the normalized fluorescence intensity of ligand-bound fraction of P W0100788 the aptamer with the ligand concentration. Nonlinear regression plot has been used to calculate the binding affinity of the ligand with the aptamer. Aptamer 13 showed specific binding to AFM1, with a binding affinity (KD) of 6.7±1.7 nM (FIG.8). However, it did not interact with other molecules like AFB1 and ZEA (Figure 2B-E). The KD of Aptamer 13 was found to be the lowest as compared to other aptamers selected.

[0193] A gold electrode was employed for the conjugation of a thiol-modified aptamer, exploiting the strong chemisorption between thiol groups and the gold surface to form a stable self-assembled monolayer. This immobilization strategy ensures robust attachment of the aptamer on the electrode, which is critical for sensitive detection. Under optimized conditions — aptamer concentration of 4 pM, coating time of 4 hours, and aptamer-target incubation time of 20 minutes (Pankaj et al., 2022) — the addition of AFM1 resulted in a significant decrease in impedance. This decrease is attributed to conformational changes or altered surface properties upon target binding, facilitating electron transfer at the electrode interface. FIG.9A shows Nyquist plot of EIS measurements with increasing concentrations of AFM1, plotted for detection of AFM1. FIG.9B shows the charge-transfer resistance (Ret) fitted against the concentrations of AFM1 to plot a linear graph for EOD calculation in buffer. The aptasensor exhibited a clear concentration-dependent decrease in impedance when tested with AFM1- spiked samples (FIG.9), demonstrating its quantitative sensing capability. The biosensor showed a strong linear correlation (high R2value) between AFM1 concentration and impedance response. Importantly, the limit of detection was as low as 5.32 fg / mE in buffer, highlighting the sensor’s high sensitivity for detecting trace levels of AFM1.

[0194] Further, the cross -reactivity of AFM1 with other major mycotoxins was evaluated, including Aflatoxin B 1 and Zearalenone (ZEA). Notably, no cross -reactivity was observed, as confirmed by MST analysis. In addition, AFM1 was spiked into biological fluids at varying concentrations ranging from 0.1 to 10 fm / mL. The aptamer remained stable under these conditions, showed no cross -reactivity, and successfully detected AFM1 across the tested concentration range.

[0195] Electrochemical aptasensors offer a highly sensitive and rapid platform for the detection of aflatoxin Ml (AFM1) in milk and dairy products. These sensors utilize aptamers, and detect low detection limits, often reaching femtomolar or pg / mL levels, enable early contamination monitoring. Electrochemical aptasensors, in particular, provide excellent selectivity, portability, and compatibility with point-of-care systems. Due to their label-free, cost-effective, and real-time detection capabilities, these sensors are promising tools for ensuring food safety and regulatory compliance in dairy screening applications. P W0100788

[0196] ADVANTAGES OF THE INVENTION i. Aptamer based technology offers several advantages for the detection of Aflatoxin Ml (AFM1), a potent mycotoxin produced by fungi. This technology combines the specificity of antibodies with the versatility of aptamers, creating a powerful tool for sensitive and selective AFM1 detection. ii. Unlike traditional antibody -based methods, aptamers are single-stranded DNA or RNA molecules that can be generated through the systematic evolution of ligands by exponential enrichment (SELEX). Aptamers has ability to bind target molecules with high specificity and affinity, similar to antibodies. iii. In the case of AFM1, aptamers can be selected to recognize and bind specifically to the toxin’s unique molecular structure, ensuring accurate detection even in complex matrices such as food samples. iv. The immunoaffmity aspect of this technology involves immobilizing the AFMl-specific aptamer onto an immune-affinity column. This column acts as a selective trap for AFM1, enabling efficient extraction from complex sample matrices. Compared to traditional immunoaffmity columns that use antibodies, aptamer-based columns offer advantages like higher stability, ease of synthesis, and cost-effectiveness. v. Additionally, aptamers can be easily synthesized, allowing for forthright production and modification to enhance binding properties. This flexibility is crucial for tailoring the aptamer’s characteristics to optimize AFM1 detection, such as altering the length or sequence to improve affinity. Moreover, aptamers are less susceptible to batch-to-batch variability that can sometimes affect the performance of antibody-based assays, ensuring consistent and reliable results. vi. The versatility of aptamers extends to the detection platform itself. Aptamer based technology can be integrated into various detection methods, including electrochemical, fluorescence, and surface plasmon resonance (SPR) techniques. This adaptability enables the development of portable and user-friendly AFM1 detection devices suitable for both laboratory and field applications. The signal amplification strategies commonly used in aptamer-based assays further enhance sensitivity, allowing for the detection of low AFM1 concentrations in diverse sample types.

Claims

P W0100788We claim:

1. An aptamer having specific binding affinity to Aflatoxin Ml(AFMl), wherein the aptamer comprises a nucleotide sequence having SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence having 80%-99% sequence identity or a functional fragment thereof.

2. The aptamer as claimed in claim 1, wherein the aptamer is a single-stranded DNA (ssDNA) or a single-stranded RNA (ssRNA).

3. A process for the preparation of an aptamer having specific binding affinity to AFM1, wherein the process comprising the steps of(a) adding AFM1 mycotoxin stock solution to an immune affinity column (IAC);(b) adding an ssDNA library solution to immune affinity column (IAC) of step (a) followed by incubating for a specified period under specified conditions;(c) eluting the IAC with nuclease free water or a suitable buffer to obtain the bound oligonucleotides:(d) amplifying the bound oligonucleotides as obtained in step (c) by polymerase chain reaction (PCR) using suitable forward and reverse primers to obtain amplicon;(e) subjecting the amplicon as obtained in step (d) to several iterations of positive SELEX in the presence of Aflatoxin Ml mycotoxin;(f) subjecting the amplicon of step (e) to a number of iterations of negative SELEX against mycotoxins other than aflatoxin Ml (AFM1);(g) digesting the dsDNA amplicon of steps (e) & (f) using suitable exonuclease at specified conditions;(h) cloning and transforming the Aptamers to obtain the ssDNA Aptamers having Seq ID NO: 1-16.

4. The process as claimed in claim 3, wherein the process comprises the steps carried out for a specified period under specified conditions comprising:(a) adding 15-25 pL AFM1 mycotoxin stock solution (l-100ng / ml) to an immune affinity column (IAC);(b) adding 70-120 pL ssDNA library solution (10-100 ng / mL) to said immune affinity column (IAC) and incubating for a period of 30 minutes to 1.5 hours at a temperature of 23-25°C;P W0100788(c) eluting the immune affinity column (IAC) with nuclease free water to obtain the bound oligonucleotides:(d) amplifying the eluent of step (c) by polymerase chain reaction (PCR) having nonbiotin labelled forward primer (5’- ATGCGGATCCCGCGC3’), and -biotinylated reverse primer (5’ Bio-GCGCAAGCTTCGCGC-3’);(e) subjecting the amplicon of step (d) to seven-eight iterations of positive SELEX in the presence of Aflatoxin Ml mycotoxin;(f) subjecting the amplicon of step (e) to three-five iterations of negative SELEX against the zearalenone (ZEA), citrinin (CIT), and Aflatoxin Bl (AFB1) mycotoxins;(g) digesting the dsDNA amplicon of steps (e) & (f) with 10-100 U / mL of X- exonuclease enzyme at 25-40°C for 3-5 h;(h) cloning and transforming the Aptamers to obtain the ssDNA Aptamers having SEQ ID No: 1-16;(i) confirmation of the aptamer obtained in step (h) by PCR and sequencing; and(j) testing the developed aptamer obtained in step (i) against AFM1 toxin conjugate through aptamer-based Enzyme Linked Immuno Sorbent Assay (ELISA).

5. A composition comprising a plurality of aptamers specific to AFM1 , wherein said aptamers comprise nucleic acid sequences of SEQ ID. No. 1,2, 3, 4, 5, 6, 7, 8, 9,10,11,12,13,14,15,16, a homologous sequence or a functional fragment thereof.

6. A device for detecting AFM1 in a sample, comprising one or more aptamers of having nucleotide sequence of SEQ ID. No. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence or a functional fragment thereof, wherein the device is a lateral flow device or lateral flow test strip or dip stick.

7. The device as claimed in claim 6, wherein the device is an electrochemical sensor device comprising a redox labelled aptamer immobilized on the surface of the electrode, wherein the detection of AFM1 in the sample to be tested is achieved by measuring the electrochemical signal generated due to AFM1 -aptamer binding.

8. A kit for the detection of AFM1 in a sample, wherein the kit comprises one or more aptamers of nucleotide sequence having SEQ ID. No. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, a homologous sequence or a functional fragment thereof, along with an aptamer-functional substance complex, buffer solution, binding buffer, chromogenic dyes and instruction manual.

9. A method for the detection of AFM1 in a sample, the method comprising the steps of:P W0100788(i) providing a sample suspected of containing Aflatoxin Ml;(ii) conducting a detection assay utilizing single- stranded DNA (ssDNA) aptamers specific to Aflatoxin Ml as claimed in claim 1, followed by determining the presence or absence of Aflatoxin Ml in said sample based on the binding affinity of said aptamers.

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