WO2023199350A1

WO2023199350A1 - Lateral flow assay strip with rationally designed peptides for individual or simultaneous analysis of mycotoxins

Info

Publication number: WO2023199350A1
Application number: PCT/IN2023/050351
Authority: WO
Inventors: Vandana GHORMADE
Original assignee: Macs- Agharkar Research Institute
Priority date: 2022-04-11
Filing date: 2023-04-11
Publication date: 2023-10-19

Abstract

The present invention relates to a lateral flow assay strip encompassing rationally designed short peptides as recognition molecules, its process of development and their utility in individual and multiplexed detection of multiple mycotoxins from fungus-infected food and feed using short peptides in a paper-based format. The POC strip comprises sample site for applying the sample with one or more members of mycotoxin analytes particularly Aflatoxin, Ochratoxin alone or in combination; a conjugated pad site, pre-treated and loaded with AuNPs- biotinylated AFB1 peptide and AuNPs-biotinylated OTA peptide (pooled together loaded at one site), a nitrocellulose membrane sheet loaded with Protein A as a control line, AFB1 and OTA separately as a test line, an Absorption pad to collect excess waste. The POC strip enables detection of microbially contaminated food products particularly cereals, processed cereals, fruits with high dextrose content, agricultural product particularly wheat, peanut, grapes, animal feed particularly poultry feed, dog feed.

Description

TITLE

Lateral flow assay strip with rationally designed peptides for individual or simultaneous analysis of mycotoxins

FIELD OF THE INVENTION

The present invention relates to a lateral flow assay strip encompassing rationally designed short peptides as recognition molecules, its process of development and their utility in detection, individual as well as multiplexed mycotoxins from fungus-infected food and feed. More particularly, the present invention relates to a designing of peptides for individual as well as simultaneous detection of mycotoxins wherein the presence of mycotoxins is carried out using short peptides in a paper-based format.

Furthermore, the synthetic peptides are designed on the basis of the mycotoxin affinity that provides an easy replacement for expensive and labile antibodies which can be employed in lateral flow assay for multiplexed detection of Aflatoxin and Ochratoxin. Therefore, the instant invention relates to a rapid, cheap and on-site Paper based mycotoxin detection assay which is independent of any equipment. Furthermore, using the rationally designed peptides for specific detection of AflatoxinBl and Ochratoxin A enables the multiplexed single platform detection.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF THE PRIOR-ART

Mycotoxins are secondary metabolites produced by fungi that often contaminate food and feed. Fungal species belonging to Aspergillus and Penicillium genera infect crops in the field or during storage leading to mycotoxin contamination of foods and feed, globally (Rodrigues and Naehrer, 2012). Mycotoxins are recalcitrant to degradation by heat or cooking. Mycotoxin contamination leads to abdominal pain, hepatotoxicity, nephrotoxicity, and immune-suppression and also contributes to loss of human life (Wood et al 2003, Bryden 2007). The Food and Agriculture Organization have estimated that 25% of the world’s food crops are significantly contaminated with mycotoxins (WHO, 1999). The Center for Disease Control, US has estimated that 4.5 billion people are exposed chronically to mycotoxins, with 40% from the developing countries (Speight, 2012). Beyond their health impacts, mycotoxins lead to yearly trade losses ~$1.2 billion in USA. The Hazard Analysis and Critical Control Point (HACCP) approach proposes the testing of food matrices at each stage of production, storage, retailing and distribution. The increasing consumer awareness, rise in global trade and compliance for trade require on-site mycotoxin detection tools that can simplify the testing for the large amounts of food to be tested.

The main efforts in the global control of Ochratoxin and Aflatoxin are directed towards species of Aspergillus and Penicillium (Duarte et al., 2010). Mycotoxins are often produced when the fungus is under stress, when the temperature, water activity or amount of oxygen conditions are altered (Magan et al 2010). In EU, the permissible limit for Aflatoxin Bl (AFB1) is 4ug/kg and OchratoxinA (OTA) is 5ug/kg (European mycotoxin awareness network, Feb 2012). The EU permissible level for Aflatoxin and Ochratoxin for processed cereal-based foods for infants and young children are 0.025ug/kg and 0.5 ug/kg, respectively. In India, a limit of 20 pg/kg of OTA and 15ug/kg AFB1 has been defined by the Food Safety and Standards Authority of India in wheat and wheat based products.

In India, analysis of wheat samples showed 58% (29 samples) with 1.36 - 21.17 pg/kg OTA contamination, including 26% (13 samples) exceeding 5 pg/kg level as described by EU (Kumar et al 2012). It was reported that OTA intake in adult population saturated the provisional tolerable weekly intake (PTWI) by 0.97-2.49 folds. Contamination in black pepper spice samples was reported by Ramesh and Jaygoudar (2014) to show that OTA content < 5. 0 - 5.9pg/Kg within the EU consumption limit. Jeswal and Kumar (2015) showed that high amount of Aflatoxin was detected in red chilli (219.6 ng/g) while OTA was detected in black pepper (154.1 ng/g). Occurrence of OTA was reported common herbal medicines such as Holarrhena antidyscentrica (1.14 - 2.34 pg/g) bark and Taccaaspera (0.3 - 0.74 pg/g) rhizomes was associated with isolation of toxigenic Aspergillus ochraceus, A. sulphureus and Penicillium viridicatum fungi were isolated from these samples. Devi et al (2002) reported 38% Aflatoxin and 6% Ochratoxin A contamination in 216 chicken poultry feed ingredients with co-occurrence of AFB 1 and OTA contamination in groundnut, sorghum, sunflower, rice bran, and millet samples. AFB1 was reported in maize and mixed feeds and soybean samples. Magan et al. (2011) suggested that climate change toward hot temperatures and drought could increase the risk of migration of spoilage organisms and pathogens.

There exist different classical methods that are commonly used for quantitative determination of mycotoxins, including High Performance Liquid Chromatography (HPLC) coupled with ultraviolet (UV), liquid chromatography/mass spectrometry (LC/MS), gas chromatography coupled with electron capture detection and thin- layer chromatography (TLC). In addition, Enzyme-Linked Immunosorbent Assays (ELISA) or membrane-based immunoassays are frequently used commercially for screening purposes (Bondy and Pestka, 2000; Ono et al., 2000; Alakonya et al., 2008). Many commercial ELISA kits and immunoaffinity columns are available for mycotoxin determination abroad. In India, ICRISAT has developed an ELISA kit for the determination of Aflatoxin. Although PCR based methods can detect the presence of spoilage organism it is not correlated to the mycotoxin production by the organism. The Codex Alimentarius (CAC) includes the determination of mycotoxin levels in Good Agricultural Practice to reduce contamination in cereals (CAC/ RCP 51, CAC, 2003). As the control of mycotoxin contamination is difficult the continuous monitoring calls for a sensitive, newer, cost-effective, simple methodology to quantify mycotoxin producers and mycotoxins.

Detection of mycotoxin is routinely carried out with HPLC or ELISA methods which are expensive, require sophisticated equipment and skilled personnel and are not portable. Paper based assays are sensitive, simple, easy and rapid and onsite devices that can detect target analyte presence in a sample without the need for specialized and costly equipment. The method uses low cost test devices consisting of conjugation pad, membrane, sample pad and absorbent pad in either dot blot or lateral flow assay. The detection maybe based upon competitive or direct formats using sensitive recognition molecules that are mostly antibodies [Moon 2013]. The VICAM Ochra-V AQUA strip test (Massachusetts, USA) and R-BIOPHARM Ochracard (Darmstadt, Germany) for OTA detection are monoclonal antibody based tests. Most LFAs that are commercialized or cited in literature are based on recognition of toxin by antibodies (Kolosova et al 2007, Chen et al 2016). Liu et al., (2018) developed an antibody based LFA for rapid detection of OTA in red wine with LOD of lOng/mL. Antibodies are expensive and are difficult to raise against the low molecular weight toxin molecules. They are susceptible to degradation by variation in temperature, pH, or storage conditions (Tothill, et al., 2010; Heurich, et al., 2013).

Antibodies are expensive and it is difficult to raise against the low molecular weight mycotoxins. Chen et al 2020 reported the visual detection by monoclonal antibody based dot immunoassay with a cut-off limit of 50pg/kg in rice and oat samples. LFA using monoclonal antibody was developed for detection of OTA from red wine with cut-off limit of lOng/mL (Liu et al. 2018). Sun et al 2017 reported nanobody based competitive dot ELISA for detection of OTA in cereals, the cut-off was 5pg/kg. Nan body-alkaline phosphatase fusion protein was used in dot blot assays with LOD 5pg/kg (Tang et al 2018).

As compared to antibodies that are commonly used in immune-sensors, short chain peptides present several advantages such as ease of synthetic synthesis, avoiding animal usage, molecular stability and availability. Using specific peptides as the recognition molecule in lateral flow assay technology could provide a promising approach for semi-quantitative, rapid, easy and cost-effective mycotoxin detection. Previously, Ochratoxin binding peptides were identified for use in affinity columns and mycotoxin separation [Bazin et al 2013, Giraudi et al 2007].

Reference may be made to WO2017197914A1, which relates to an antigenaptamer-based competitive assay test strip, its application in testing an Aflatoxin Bl or ML The test strip comprises of a sample absorption pad, a marker pad, a reaction film, a water absorbent pad, and a base plate. The marker pad is coated with a detection probe which is an aptamer labelled with a detection target for displaying an indicator of signal intensity. The reaction membrane is provided with a detection zone and a quality inspection zone. The detection zone is fixedly coated with a target antigen. The quality control zone is fixedly coated with a quality control probe. The target antigen is a conjugate of the inorganic small molecule target and a carrier protein. So, the detection zone is coated with a conjugate of Aflatoxin Bl hapten and a carrier protein. The carrier protein includes casein, bovine serum albumin or chicken ovalbumin. The indicator for indicating signal intensity comprises a fluorescent substance, biotin, radioisotope, electron-dense substance, colloidal gold or Enzyme. The nucleic acid sequence of the quality control probe is complementary to or linked to the aptamer of the complementary sequences. The sequence to which the aptamer is linked is PolyT18. Application of the disclosed test strip is detection of Aflatoxin Bl and Ml and Thrombin as food contaminants, clinical disease markers.

Reference may be made to US20090246340A1, which relates to the synthetic ligands, particularly peptides, able to bind to the mycotoxin i.e. Ochratoxin A and the uses thereof for the determination and the separation of the Ochratoxin A from a food matrix, preferably wine. The binding system comprises of at least a peptide selected from the sequences SEQ ID NO.: 1-9, at least a spacer arm particularly amino butyric acid conjugated with said peptide. Solid phase is selected from chromatography resin, micro titre plate, micro titre plate with filter septa, membrane, strip, sensor surface. The process for the detection of Ochratoxin involves steps as: i) arranging a solid phase including at least a binding system; ii) contacting said sample with said solid phase; iii) detecting the bond between said binding system with Ochratoxin A existing in said sample. The combinatorial libraries are prepared for an amino acid sequence which shows evident molecular recognition properties towards the Ochratoxin A. The amino butyric acid used as a spacer arm due to having per se an amino acidic form.

Reference may be made to US Patent, US2003203412A1, which relates to a biochip and a kit for detection of small molecules like Aflatoxin. The biochip of the present invention comprises of a solid support and carrier- linked small molecules immobilized onto the solid support. The carrier is a protein selected from the group consisting of human serum albumin (HSA). The biochip further comprises of a blank control, a negative control, a sample preparation control, an immobilization control, and a data normalization control immobilized on the surface of the solid support. The solid support is of selected from the group consisting of ceramic, glass, silica, quartz, nylon, plastic, polystyrene, nitrocellulose, and metal. The binding molecule is an antibody or a polymer and a label. The label is a molecule selected from the group consisting of a fluorescent, an enzymatic, a biotin, a radioactive, and a luminescent label. The detection mechanism is detecting the presence or absence or quantity of the label on the biochip remaining after binding of the binding molecule to the small molecule compound in the conjugate immobilized on the surface of the biochip. The application of the biochip is for doping agents testing.

Reference may be made to Patent CN108794580A, which relates to Aflatoxin - enzyme-linked immunosorbent assay device based on nano-antibody and antigen analog peptide. The peptide mimic of Aflatoxin is utilized in the assay to reduce the direct usage of the toxin in the assay. The mimic only replaces Aflatoxin and does not recognize it. The presence of Aflatoxin is detected using the peptide mimic of Aflatoxin in a competitive format with the nano -antibody.

A rational approach for designing peptide recognition molecules is utilized by identifying the target molecules of these toxins and studying the toxin-receptor interaction. Simulation of the binding of the toxin and receptor molecules is utilized to identify the amino-acid interactions. Such amino acids are then employed in designing the peptide followed by addition of linker amino-acid sequences to overcome possible steric hindrance during the toxin-receptor complex formation. Finally, these peptides are decorated with a biotin moiety to enhance the binding to the streptavidin bearing indicative nanoparticles labels.

Using such peptides as the recognition molecule in lateral flow assay technology could provide a promising approach for semi-quantitative, rapid, easy and cost- effective mycotoxin detection. Peptides have various advantages in terms of molecular stability and availability, compared to antibodies, which are being more commonly used in immuno sensors. Using specific peptides as the recognition molecule in lateral flow assay technology could provide a promising approach for semi-quantitative, rapid, easy and cost-effective mycotoxin detection. Therefore, the use of modified short peptides is proposed as recognition molecules for detection of mycotoxins in a paper-based lateral flow assay. The development of multiplexed diagnostic assays for rapid detection of mycotoxins is the need for proper food safety in a large country like India.

In view of the hitherto reported prior-art, we understand that, there exist various literatures that have reported the usage of HPLC and ELISA for the detection of mycotoxins in a given sample. However, these methods are based on HPLC, immune-affinity columns that detect single mycotoxin at a time and are time consuming and equipment intensive. Serological ELISA assays which may be multiplexed are mono -/poly-clonal antibody based and is expensive. Thus, raising antibodies towards small molecule like mycotoxins are difficult and also face issues of toxicity. Proteinaceous nature of antibody is affected by the organic solvents employed for toxin extraction. Specific peptides as stable, sensitive, affordable recognition agents of mycotoxin can have good utility improving the diagnostic sensitivity of portable, on-site paper based assays.

Thus, in view of the drawbacks reported in the above prior-arts, there exists a dire need to for development of Point of care (POC) testing device encompassing rationally designed peptides for specific recognition and multiplexed detection of mycotoxin contaminants in a given sample. Therefore, the instant invention relates to a simple and inexpensive Point of care (POC) testing strip, its process of development and method for rationally designing peptides for specific recognition and multiplexed detection of mycotoxin contaminants in a given sample such as food and feed.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to provide a POC test strip for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin A, Ochratoxin or its combination. Another objective of present invention is to provide a POC test strip for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin A, Ochratoxin or its combination by lateral flow assay method.

Yet another object of present invention is to provide simple and on-field detection test strip encompassing rationally designed peptides which are specific for individual and multiplexed detection of mycotoxin contaminants in a given sample.

Another objective of the present invention is to provide a rapid, visual and on-site paper based mycotoxin detection which is independent of any equipment.

Yet another objective of the present invention is to provide a POC test strip encompassing the rationally designed recognition peptides for specific detection of Aflatoxin Bl and Ochratoxin A individually or in combination.

Another object of present invention is develop rapid test system for determination of contaminants such as mycotoxins in food previous to or during production, based on easy-to-handle immunoassays with naked eye detection.

Another object of present invention is to provide field usable kit comprising of POC test strip with the limit of detection up to 175 ng/ml and detection time up to 20-25min.

Another objective of present invention is to provide field usable kit which is highly specific yet inexpensive and easy to mass-produce, store and implement.

SUMMARY OF THE INVENTION

The present invention deploys usage of a POC test strip (100) encompassing synthetic peptides designed on basis of the mycotoxin affinity, employed in lateral flow assay to provide an easy replacement for expensive and labile antibodies for multiplexed detection of Aflatoxin B 1 and Ochratoxin.

According to one embodiment of present invention, POC test strip (100) for multiplexed detection of Aflatoxin and Ochratoxin comprises of a sample site (101) for applying the sample with one or more members of mycotoxin analytes particularly Aflatoxin, Ochratoxin alone or in combination, a conjugated pad site (102) which is pre-treated and loaded with pooled gold nanoparticle coated biotin tagged short peptides of sequences (103, 104), particularly Seq ID 1 and Seq ID 2 (pooled together and loaded on conjugate pad), a nitrocellulose membrane sheet (105) of pore size of 8pm pore and 25mm, loaded with Protein A as a control line, and OTA specific peptide and AFB1 specific peptide separately as a test line 1 and 2 respectively, an absorption pad (106) to collect excess waste and to ensure no back flow of the fluid.

In an embodiment of the invention, the process for development of the POC test strip for multiplexed detection of Aflatoxin and Ochratoxin comprises of the following steps:

1. Rational designing of specific peptide Seq ID 1 towards Ochratoxin A and its modification by addition of a linker sequence and biotin at the N terminal (Biotin-KSGSFNHPK) for Ochratoxin A detection.

2. Rationally designing of specific peptide Seq ID 2 towards Aflatoxin Bl and its modification by addition of a linker sequence and biotin at the N terminal (Biotin-KSGKSKPR) for aflatoxinBl detection;

3. Coating of AflatoxinBl and Ochratoxin A separately on test line 1 and test line 2, respectively on a nitrocellulose paper/membrane with a printer and printing the control line for protein A; and

4. Coating the gold nanoparticles with streptavidin and conjugating to the biotin tagged short peptides towards OTA and AFB 1 separately;

5. Pre-treating of conjugated pad with blocking buffer (borate buffer pH 8, skimmed milk 0.05%, Tween 0.25%, sucrose 1%), and during at 37°C overnight.

6. Pooling the OTA peptide- AuNPs and AFBl-peptide AuNPs together

7. Loading the pre-treated conjugate pad with (0.8 OD) AuNPs-AFBl peptide and AuNPs-OTA peptide and dried at RT for 2 hrs. All the membranes were assembled band cut in 4mm. 8. Running of the assay by adding PBS running buffer (0.5x) with different concentration of standard OTA and AFB1 and washing after 15-20 mins with PBST.

According to one embodiment of present invention, the short peptide sequences (103, 104) with Seq ID 1 and Seq ID 2 are designed by the computational modelling on the target proteins which shows high affinity and specificity for mycotoxin binding. The selected target proteins (107, 108) are Human serum albumin (HSA) and Phenylalanyl- Tma synthetase (PheRS) utilizing the literature knowledge about high binding affinity of mycotoxin molecules particularly AFB 1 and OTA with the HSA and PheRS, respectively (Poor et al., 2017; Ha, 2015).

The docking studies using AutoDock Vina are conducted for selection of peptide sequence specific for AFB1 and OTA from the protein structure of the Human serum albumin (HSA) and Phenylalanyl- tRNA synthetase (PheRS). The selected peptide sequences are further modified by replacing the amino acid for optimum binding energy, H-bonding, hydrophobicity and solubility. The modified peptide sequence is further screened by molecular docking on the Human serum albumin (HSA) and Phenylalanyl- tRNA synthetase (PheRS) to design the short peptides of Seq Id 1 and 2 (103, 104) with optimum characters for detection of Aflatoxin B 1 and Ochratoxin.

According to another embodiment of present invention, the linker (109) is selected as a non interacting tripeptide chain to facilitate the stereo-chemical binding between the mycotoxin analyte and the streptavidin coated gold nanoparticles (110). The selection of linker sequence depends on its contribution to optimum binding energy, H-bonding, hydrophobicity and solubility. The selected linker sequence comprises of atleast three non-interacting amino acid, particularly Lysine, Serine and Glycine.

According to another embodiment of present invention, the linker modified designed peptides having affinity for binding to AFB1 and OTA are tagged with biotin molecule at N- terminal of the linker to enhance the binding to the streptavidin bearing indicative gold nanoparticles labels by contacting it with biotin molecule. The biotin tagged linker modified Short peptides of Seq ID 1 and 2 are evaluated for binding affinity with OTA and AFB1 by performing indirect ELISA. The apparent dissociation constant (Kdapp) is calculated from the slope of regression curve obtained by plotting the fraction of bound peptide and the molar concentration of OTA and is found to be 817 nM. The apparent dissociation constant (Kdapp) is calculated from the slope of regression curve obtained by plotting the fraction of bound peptide and the molar concentration of OTA and is found to be 323 nM.

According to another embodiment of present invention, the BIOTIN - KSGSFNHPK peptide is able to detect OTA standard solution in ELISA which is in accordance with the detection of 10 ng/mL OTA by the peptide based ELISA. Similarly BIOTIN-KSGKSKPR peptide is able to detect AFB1 standard solution in ELISA which is in accordance with the detection of 10 ng/mL AFB1 by the peptide based ELISA. The ELISA test is conducted to confirm the use of biotin tagged linker modified rationally designed peptides for detection of OTA and AFB1, The designed biotinylated peptides BIOTIN-KSGKSKPR and BIOTIN- KSGSFNHPK are tested for binding to the OTA and AFB1, respectively by coating 96 well plates with different dilutions (10 ng/mL, 20 ng/mL, 40 ng/mL, 60 ng/mL 80 ng/mL, 100 ng/mL) of OTA or AFB1 in 475 mM carbonate buffer pH 9.6 in humidified container at 4°C overnight. The incubation phase is followed by washing with phosphate buffer solution and addition of streptavidin-HRP complex and again incubation.

Yet another embodiment of present invention, BIOTIN-KSGKSKPR peptide and BIOTIN-KSGSFNHPK peptide are conjugated with gold nanoparticles coated with streptavidin to construct the short peptides (103, 104), particularly of sequence Seq ID 1 and Seq ID 2. The synthesized short peptides (103, 104), particularly of sequence Seq ID 1 and Seq ID 2 are loaded on the pre-treated conjugate pad site (102).

Yet another embodiment of present invention, the short peptides (103, 104), particularly of sequence Seq ID 1 and Seq ID 2 do not show any cross reactivity up to 500 and 400 ng/mL for AFB1 specific peptide having Seq Id 1 and up to 400 and 300 ng/mL for OTA specific peptide having Seq Id 2 with complementary mycotoxins i.e. AFB1 and OTA as well as with another cooccurring mycotoxin CIT (Citrinin).

According to another embodiment of present invention, the process for the analysis of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin by the POC test strips comprises steps of: a. Preparing the sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin by diluting with 0.5X Phosphate buffer solution b. Adding the prepared sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin to the sample site (100 pl) c. Passing of the sample to the conjugated pad site and nitrocellulose membrane sheet and absorption pad d. Visualizing the color change from to this at test line 1, test line 2, control line due to reaction of AuNPs-biotinylated AFB1 peptide and AuNPs-biotinylated OTA peptide with AFB1 and OTA and protein A present the test sample respectively

Yet another embodiment of the instant invention is the permissible level of the POC test strip for multiplexed detection of Aflatoxin and Ochratoxin is 175ng/ml or less and the detection time is 25-30min.

Yet another embodiment of present invention, the kit for detection of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin, individually or simultaneously comprises of at least one POC test strips; a sealable plastic tube containing a phosphate buffer saline (0.5x), a dropper characterized to aspire the prepared sample.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 represents a schematic diagram for the POC test strip for simultaneous analysis of a sample containing one or more members of mycotoxin analytes igure 2 a detection result interpretations for competitive Lateral flow assay a) Positive result for AFB1 and OTA for samples > 175 ng/ml. b and c ) Negative tests below < 175 ng/ml d) Negative test with buffer only

Figure 3 represents pictorial formula for i) Biotinylated Short peptide of Seq ID 1 and ii) Biotinylated Short peptide of Seq ID 2

Figure 4 represent the flow chart for designing of AuNP coated-biotin tagged- Linker modified- short peptide with Seq ID 1 and 2

Figure 5A represents a schematic diagram of the docking pose of designed peptide KSGSFNHPK with OTA

Figure 5B represents a schematic diagram Docking pose of KSGKSKPR peptide with AFB 1

Figure 6A represents determination of Kd for OTA with Biotin-KSGSFNHPK peptide by slope of regression curve. B Scatchard Plot

Figure 6B represents determination of Kd for AFB1 with Biotin- KSGKSKPR peptide by slope of regression curve. B Scatchard Plot

Figure 7A represents ELISA for Standard OTA at different concentrations of 100, 80, 40, 20, lOng/mL. The values are the mean of three independent experiments and the error bar represents the standard deviation.

Figure 7B ELISA of Standard AFB1 at different concentrations of 100, 80, 40, 20, lOng/mL. The values are the mean of three independent experiments and the error bar represents the standard deviation

Figure 8A represents saturation curve for conjugation of biotinylated peptide with AuNPs

Figure 8B represents UV-Vis spectrum shift assay to confirm the conjugation of biotinylated peptide with AuNPs

Figure 9 represents absence of cross-reactivity with citrinin at 200 ng/ml (A) and 100 ng/ml (B) as compared to buffer control (C)

Figure 10 represents Reverse phase HPLC for simultaneously detectionofAFBlandOTA. A) AFB1 detected at retention time of 6.5min and OTA detected at retention time of 9min. B) Standard graph forAFBl (C) Standard graph for OTA Table No. 1: Legend and Legend Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a lateral flow assay strip encompassing rationally designed short peptides as recognition molecules, its process of development and their utility in individual and multiplexed detection of multiple mycotoxins from fungus-infected food and feed. More particularly, the present invention relates to designing of peptides for simultaneous detection of mycotoxins wherein the presence of mycotoxins is carried out using short peptides in a paper-based format.

Furthermore, the synthetic peptides are designed on the basis of the mycotoxin affinity that provides an easy replacement for expensive and labile antibodies which can be employed in lateral flow assay for multiplexed detection of Aflatoxin and Ochratoxin. Therefore, the instant invention relates to a rapid, cheap and on-site Paper based mycotoxin detection assay which is independent of any equipment. Furthermore, using the rationally designed peptides for specific detection of AflatoxinBl and Ochratoxin A is that it enables the multiplexed single platform detection. In an embodiment of present invention, Figure 1 represents a schematic diagram of the POC test strip for analysis of a sample containing one or more members of mycotoxin analytes, particularly AFB1 and OTA individually or simultaneously. The POC test strip comprises of a sample site (101) GFB-R7L (0.6), for applying the sample with one or more members of mycotoxin analytes particularly Aflatoxin Bl, Ochratoxin alone or in combination, a conjugated pad site (102) of Conjugate Release Matrix Pad PT -R5 which is pre-treated and loaded with pooled short peptide sequences (103, 104), particularly Seq ID 1 and Seq ID 2 (pooled together loaded at one site), a nitrocellulose membrane sheet (105) CNPF-SN12- L2-P25 of pore size of 8pm pore and 25mm, loaded with Protein A as a control line, AFB 1 specific peptide and OTA specific peptide separately as a test line 1 and 2 respectively, an absorption pad (106) AP080 to collect excess waste and to ensure no back flow of the fluid. The conjugated pad is pre-treated by blocking with blocking buffer (borate buffer pH 8, skimmed milk 0.05%, Tween 0.25%, sucrose 1%), and dried at 37°C overnight.

In an embodiment of present invention, Figure 2 represents a detection result interpretation for a) Positive result for AFB1 b) Positive test for OTA c) Positive test for AFB1 and OTA d) Negative test. The POC detection strip is based on competitive lateral flow assay wherein AuNPs-short peptide of seq ID 1 and 2 are coupled at the pre-treated conjugated site and the OTA and AFB1 analytes are loaded at the Nitrocellulose membrane as test line 1 and 2 respectively along with control line of Protein A. In case of positive test for AFB1 as depicted on Figure 2 (a), no color development is seen at Test line (T2, designated for AFB1) and red color development is seen at the test line 1 (T1 designated for OTA) and control line (C designated for Protein A). As the sample contain AFB1 analyte, it reacts with AuNP-Short peptide specific for the AFB 1 mycotoxin with Seq id 2 loaded at the Conjugated site (102). This results in unavailability of AuNP-Short peptide specific for the AFB1 mycotoxin with Seq id 2 for coupling with AFB1 mycotoxin present at the Test line (T2) and no red color development indicting presence of AFB1. In case of positive test for OTA as depicted on Figure 2 (b), no color development is seen at Test line (Tl, designated for OTA) and red color development is seen at the test line 1 (T2 designated for AFB1) and control line (C designated for Protein A). As the sample contains OTA analyte, it reacts with AuNP-Short peptide specific for the OTA mycotoxin with Seq id 1 loaded at the Conjugated site (102). This results in, unavailability of AuNP-Short peptide specific for the OTA mycotoxin with Seq id 1 for coupling with OTA mycotoxin present at the Test lien (Tl) and no red color development indicting presence of OTA. In case of positive test for AFB1 and OTA as depicted on Figure 2 (c), no color development is seen at Test line (Tl, designated for OTA) and at the test line 1 (T2 designated for AFB1) and red color development is seen at control line (C designated for Protein A). As the sample contains AFB1 and OTA analyte, it reacts with AuNP-Short peptide specific for the AFB 1 mycotoxin with Seq id 2 and AuNP-Short peptide specific for the OTA mycotoxin with Seq id 1 loaded at the Conjugated site (102). This results in, unavailability of AuNP-Short peptide specific for the AFB 1 mycotoxin with Seq id 2 and AuNP-Short peptide specific for the OTA mycotoxin with Seq id 1 for coupling with AFB 1 present at Test line (T2) and OTA mycotoxin present at the Test line (Tl) and no red color development indicting presence of AFB1 and OTA. In case of Negative test for AFB1 and OTA as depicted on Figure 2 (d), red color development is seen at Test line (Tl, designated for OTA) and at the test line 1 (T2 designated for AFB1) and also red color development is seen at control line (C designated for Protein A). As the sample do not contains AFB1 and OTA analyte, it doesn’t reacts with AuNP- Short peptide specific for the AFB1 mycotoxin with Seq id 2 and AuNP-Short peptide specific for the OTA mycotoxin with Seq id 1 loaded at the Conjugated site (102). This results in, availability of AuNP-Short peptide specific for the AFB1 mycotoxin with Seq id 2 and AuNP-Short peptide specific for the OTA mycotoxin with Seq id 1 for coupling with AFB 1 present at Test line (T2) and OTA mycotoxin present at the Test line (Tl) and red color development indicting absence of AFB 1 and OT A.

In an embodiment of present invention, Figure 3 represents pictorial formula for i) AuNPs-biotinylated short peptide with Seq ID 1 and ii) AuNPs-biotinylated Short peptide with Seq ID 2. AuNPs-biotinylated OTA peptide comprises of a short peptide of Seq ID 1 possessing binding affinity for Ochratoxin, a linker to permit the stereo-chemical binding with toxin and gold nanoparticles for their intended use as detection agents, a biotin, to enhance the binding of OTA specific short peptide (Seq ID 1) to the streptavidin coated gold nanoparticles, a streptavidin conjugated gold nanoparticles as a detection label. AuNPs- biotinylated AFB1 peptide comprises of a short peptide of Seq ID 2 possessing binding affinity for Aflatoxin, a linker to permit the stereo-chemical binding with toxin and gold nanoparticles for their intended use as detection agents, a biotin, to enhance the binding of AFB 1 specific short peptide (Seq ID 2) to the streptavidin coated gold nanoparticles and a streptavidin conjugated gold nanoparticles as a detection label.

In an embodiment of present invention, computational modelling approach is utilized for rational design of peptide specific for mycotoxin particularly AFB1 and OTA detection. Figure 4 depicts the flow chart for development of the gold nanoparticle coated-biotin tagged-linker modified-Short peptides with Seq ID 1 and 2. Peptide design for AFB1 and OTA is performed using bio informatics tools with the steps involving carrying out computational modelling for rational design of peptide by identifying the possible target proteins which shows high affinity and specificity for mycotoxin binding, performing Molecular docking to study the involvement of different amino acid residues involved in the mycotoxin-target binding by using peptide design molecular docking, selection of amino acid residues involved in binding of the target protein with the mycotoxin, designing of peptides and screening on basis of parameters such as their binding energy, presence of H-bonding, hydro -phobicity and solubility, addition of a linker sequence to designed peptide to permit for stereo-chemical binding with toxin and gold nanoparticles for their intended use as detection agents.

According to another embodiment of present invention, the target proteins that show specific binding to AFB1 and OTA are selected molecular docking as an interacting molecule or receptor. In accordance with literature references, Human serum albumin (HSA) is selected as interacting molecule or receptor for ligand AFB1 binding study and Phenylalanyl- Tma synthetase (PheRS) is selected as interacting molecule or receptor for ligand OTA binding study. (McMasters and Vedani 1999; Poor et al 2017). Ligand coordinates in case of AFB1 and OTA as ligand are obtained from PubChem Compound database (http ://pubchem. ncbi. nlm. nih. go v/) .

According to another embodiment of present invention, molecular docking is performed using AutoDock Vina to design the short peptide having high affinity with the OTA and AFB1 as a Ligand which can be further used in a detection of mycotoxin. Blind docking is performed impartially by covering the complete peptide region with a grid. The grid box size is set to 40 A° x 40 A° x 40 A° (x, y and z) with 1 A⁰ spacing between the grid points. During the blind docking procedure, 9 different conformers are generated for ligands particularly AFB 1 and OTA by applying parameters such as addition of H-bond and removing non-polar hydrogen while the default parameters are addition of charges, ignoring water and chains of non-standard residues. During docking, selection of the final pose of ligand bound with the protein is done by giving priority to the lowest binding energy conformation. A comprehensive analysis of residues involved in ligandprotein interaction is conducted using the Chimera Software. Molecular docking displays the involvement of AFFHWRER amino acids in the binding pocket of PheRS and KHYRSAIIRR amino acids in the binding pocket of HSA or Sudlow site I which allows the binding of OTA and AFB1 by hydrophobic interaction with the protein molecule.

According to another embodiment of present invention, further, these amino acids i.e. AFFHWRER and KHYRSAIIRR are selected for the peptide designing and subjected to virtual screening by applying molecular docking to evaluate Ligand (OTA)- receptor (PheRS) and Ligand AFB1 -receptor (peptide HSA) interaction. AFFHWRER peptide sequence displayed binding energy of -5.6 kcal/mol. No hydrogen binding with the target protein and very high hydrophobicity is seen. Similarly peptide KHYRSAIIRR shows binding energy of -4.8 kcal/mol, do not display any hydrogen binding with the target protein and very high hydrophobicity. In another embodiment of present invention, the selected peptides AFFHWRER and KHYRSAIIRR specific for OTA and AFB1 respectively are modified by amino acid replacement to increase its affinity and solubility. The peptides AFFHWRER and KHYRSAIIRR undergone couple of iteration of amino acid to arrive at amino acid sequence with low binding energy which implies to stable binding of the toxin and protein, presence of hydrogen-bonding to influence on specificity of binding and adsorption and good solubility for diagnostic power. Table 2 and Table 3 indicates list of peptides designed for OTA and AFB1 binding and their characteristics.

Table 2: List of peptides designed for ochratoxin binding and their characteristics

Table 3: List of modified peptides for aflatoxinBl binding and their characteristics

According to another embodiment of present invention, hexa-peptide SFNHPK is selected as a peptide specific for binding to the OTA mycotoxin, which is analysed in the sample as it demonstrates the presence of three hydrogen bonds, low hydrophobicity and good solubility which is mandatory for good binding with analyte. Similarly pentapeptide with amino acid sequence KSKPR is selected as a peptide specific for binding to AFB 1 mycotoxin, which is analysed in the sample as it demonstrates the presence of two hydrogen bonds, low hydrophobicity and good solubility which is mandatory for good binding with analyte.

According to another embodiment of present invention, the designed hexapeptide specific for OTA and pentapeptide specific for AFB1 is further conjugated with short chain amino acid linker molecule. The selection of linker molecule is based on non- interfering property and permitting stereo -chemical binding between toxin and gold nanoparticles for their intended use as detection agents. The linker sequences consisting of Glycine, Glycine -Glycine and Glycine-Glycine-Glycine are screened for binding studies with mycotoxin analytes, particularly OTA and AFB1. Table 4 depicts the binding characteristics of different linker sequences with OTA and Table 5 depicts the binding characteristics of different linker sequences with AFB1.

Table 4: Binding characteristics of different linker sequences with OTA as a ligand

Table 5: Binding characteristics of different linker sequences with AFB1 as a ligand

According to another embodiment of present invention, the liker modified hexapeptide specific for OTA analyte selected for further conjugation is KSGSFNHPK, as depicted in Figure 5A and the liker modified pentapeptide specific for AFB1 analyte selected for further conjugation is KSGKSKPR, as depicted in Figure 5B.

According to another embodiment of present invention, biotin tagging is done by conjugating biotin molecule to N-terminal of the liker modified Hexpeptide specific for OTA i.e. KSGSFNHPK to give Biotin-N’- KSGSFNHPK and with N- terminal of the liker modified Pentapeptide specific for AFB1 i.e. KSGKSKPR to give Biotin-N’-KSGKSKPR.

According to another embodiment of present invention, binding affinity of the biotin tagged linker modified Hexapeptide specific for the OTA i.e. Biotin-N’- KSGSFNHPK and biotin tagged linker modified pentapeptide specific for AFB1 i.e. Biotin-N’-KSGKSKPR is determined by ELISA assay and compared with binding affinity of peptides, particularly PheRS and HSA for ochratoxin and aflatoxin Bl respectively. The ELISA assay involves reaction of OTA at 100 - 2500nM concentrations in PBS (Phosphate Buffered Saline, pH 7.2) with the (BIOTIN-KSGSFNHPK) peptide (125 nM) in PBS buffer for 15 h at RT, transferring an aliquot (lOOul) of the above suspension into 96-well plate precoated with 2500 nM of OTA toxin and incubation with mild shaking at room temperature for Ih. Similarly reaction of AFB1 at 100 - 2000nM concentrations in PBS (Phosphate Buffered Saline, pH 7.2) with the (BIOTIN -KSGKSKPR) peptide (300 nM) in PBS buffer for 15 h at RT, transferring an aliquot (lOOul) of the above suspension into 96-well plate pre-coated with 200 nM of AFB1 toxin and incubation with mild shaking at room temperature for lh. Further from both the ELISA assay kits, the unbound peptide is removed by washing with 200 pl PBST buffer (PBS and 0.1% tween-20, pH 7.2). After that, the absorbance is measured at 450 nm by using Horseradish peroxidase -streptavidin and 3, 3’, 5, 5 ’-Tetramethylbenzidine (TMB) (Sigma, USA). The fraction of bound peptide is plotted against the concentration of OTA and AFB1 used. The apparent equilibrium dissociation constant (Kd, app) is calculated from the slope of regression curve. The quantitative estimation for affinity of specific peptide (KSGSFNHPK) with the OTA and KSGKSKPR with AFB1 is obtained by performing a kinetic ELISA.

According to another embodiment of present invention, the apparent dissociation constant (Kdc/pp) calculated from the slope of regression curve obtained by plotting the fraction of bound peptide and the molar concentration of OTA and AFB1 is 817 nM and 323 nM, respectively, as depicted in Figure 6A, 6B.

According to another embodiment of present invention, detection capacity of the biotin tagged linker modified Hexapeptide specific for the OTA i.e. Biotin-N’- KSGSFNHPK and biotin tagged linker modified pentapeptide specific for AFB1 i.e. Biotin-N’-KSGKSKPR is evaluated by ELISA assay by testing its binding with OTA and AFB1, respectively. ELISA is performed by coating 96 well plates with different dilutions(10ng/mL, 20ng/mL, 40ng/mL, 60ng/mL 80ng/mL, lOOng/mL) of OTA or AFB1 in 50 mM carbonate buffer pH 9.6 in humidified container at 4°C overnight. 1 pg/rnL of biotinylated peptide was added to the plate with gentle rocking for 2 h after washing five times with 200 pl PBS. Plates are washed five times by PBST (PBS and 0.1% tween-20), then streptavidin-HRP complex (100 pl of 1:10000 dilution in PBS pH 7.2) is added to the well and incubated for 1 h. The colour is developed using TMB as substrate and the absorbance is measured in 450nm. A negative control without the antigen yielded a background absorbance, which is subtracted from each well. The Biotin-N’- KSGSFNHPK peptide is able to detect OTA standard solution in ELISA at lOng/mL, as depicted in Figure 7A. The Biotin-N’-KSGKSKPR peptide is able to detect AFB1 standard solution in ELISA at lOng/mL, as depicted in Figure 7B. According to another embodiment of present invention, Colloidal gold nanoparticles (AuNPs) are synthesized according to citrate reduction method (Frens et al., 1973). The method involves adding 1.2 mL of 1% gold chloride solution (HAuC14, SRL, and India) to 100 mL boiling water, stirring magnetically, addition of 2 mL of 1.2% sodium citrate to the boiling solution, continuation of boiling till development of wine red color. The synthsized colloidal solution is allowed to cool to room temperature and stored in dark bottles at 4°C. The colloidal gold nanoparticles are in the range of 25+12 nm. From the zeta sizer the size of AuNPs is 18.08nm with PDI 0.124. The zeta potential of the AuNPs was -40.9 mV.

According to another embodiment of present invention, conjugation of biotinylated peptides Biotin-KSGSFNHPK and Biotin- KSGKSKPR with gold NPs as labels is facilitated. The biotinylated peptides are conjugated with AuNPs by streptavidin-coated AuNPs, using streptavidin-biotin interaction. First, streptavidin is conjugated to gold nanoparticles according to Hermanson, (2010). Briefly, streptavidin (1 mg/mL, Sigma, USA) is dissolved in 0.1 M sodium phosphate buffer, pH 7.4 with stirring. 50 pL of streptavidin is added to ImL of colloidal gold solution and reacted for 20 min at room temperature. The resultant mixture is centrifuged at 14,000 rpm for 10 min and the pellet is re-suspended in 0.1 M sodium phosphate buffer and stored at 4°C until use. For the preparation of conjugates (AuNPs-Streptavidin-Biotin-peptide), 30pL of the biotinylated peptide (lug/lOOul PBS, pH 7.0) is added to 500pL of a streptavidin- colloidal gold solution. After incubation at room temperature for 20 min, 20 pL of 1% skimmed milk is added in the solution and incubated for 10 min. The mixture is centrifuged at 10,000 rpm for 10 min and the supernatant is discarded carefully. The loose pellet is re-suspended in ImM sodium phosphate buffered saline, pH 7.2 and stored at 4°C until use. The conjugation AuNPs and sterpatividin is confirmed by the UV-Vis absorption spectroscopy (Figure 8A). The blue shift in the peak as compare with bare AuNPs suggested that the streptavidin is conjugated with the AuNPs. The saturation curve confirms conjugation of AuNPs- Streptavidin to peptide at 25ug concentration (Figure 8B). According to another embodiment of present invention, AuNPs-biotinylated OTA peptide and AuNPs-biotinylated AFB1 peptide are formulated as competitive Lateral Flow Assay (LFA) for multiplexed detection of OTA and AFB1, as depicted in Figure 1, 2. For the LFA, 5 mg/ml Protein A in PBS, pH 7.2 is printed as control line on an 8pm nitrocellulose membrane (NC). Further, 10 mg/ml AFB1 and OTA in PBS buffer pH 7.2 are printed as test linel (4 times) and test line2 (4 times), respectively. The NC membrane is dried at 37°C for 2 hrs. The conjugate pad (PTR5) is blocked with blocking buffer (borate buffer pH 8, skimmed milk 0.05%, Tween 0.25%, sucrose 1%), and dried at 37°C overnight. Conjugate pad is loaded with (0.8 OD) AuNPs-AFBl peptide and AuNPs-OTA peptide and dried at RT for 2 hrs. Peptide conjugated AuNPs (500uL) are centrifuged and the pellet is re-suspended in 15ul borate buffer (100 mM, pH 8.0), containing 1% sucrose, skimmed milk 0.05%, Tween 20 (0.25%). The OTA peptide- AuNPs and AFB1 -peptide AuNPs are pooled together and the mixed conjugate is loaded on approximately 1cm pre-treated conjugate pad. All the membranes are assembled band cut in 4mm strips. The assay is run by adding PBS running buffer (0.5x) with different concentration of standard OTA and AFB1 and washed after 15-20 mins with PBST.

According to another embodiment of present invention, the designed modified peptide Biotin-KSGSFNHPK and Biotin- KSGKSKPR conjugated to gold nanoparticles is able to detect presence AFB1 and OTA at concentration of 175 ng/mL and the assay time is 25-30 mins.

According to another embodiment of present invention, AuNPs-biotinylated OTA peptide do not show cross reactivity up to 400 and 300 ng/mL with CIT and AFB1. AuNPs-biotinylated AFB peptide do not show cross reactivity up to 500 and 400 ng/mL with CIT and OTA, as depicted in Figure 9.

In an embodiment of present invention, shelf life of the developed POC test strip for analysis of a sample containing one or more members of mycotoxin analytes, particularly AFB1 and OTA individually or simultaneously is evaluated by accelerated stability studies. The POC test strip is tested at 28°C and 55°C for negative control, 150 ng/ml, 200 ng/ml and 400 ng/ml (of OTA and AFB1). Accelerated shelf life is calculated as following:

Accelerated aging rate (A)= Q10 ((Te - Ta)/ 10)

The cassettes of POC test strip is packet in aluminium packs and kept at the two temperatures for days corresponding to accelerated shelf-life in months (Table 6). The presence of Tl, T2 and C lines at low concentrations < 175 ng/ml and absence of Tl, T2 and C lines at higher concentrations >175 ng/ml confirmed the proper functioning of the assay for the time intervals.

Table 6: Accelerated Shelf life of Multiplex LFA for Mycotoxins

According another embodiment of present invention, the kit for detection of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin, individually or simultaneously comprises of at least one POC test strips, a sealable plastic tube containing a phosphate buffer saline (0.5x), a dropper characterized to aspire the prepared sample.

According another embodiment of present invention, the POC test strip and the kit for simultaneous analysis is used for qualitative analysis of one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously from the sample such as microbially contaminated food products particularly cereals, processed cereals, fruits with high dextrose content, agricultural product particularly (wheat, peanut, grapes), animal feed particularly poultry feed, dog feed.

According another embodiment of present invention, process of preparation for samples involves corse grounding samples, mycotoxin extraction, with 25 mL of acetonitrile-water (60/40, v/v) with shaking for lh, centrifugation at 5500 g, 30 min the supernatant, collection of organic phase, drying, solubilising dried sample using 1 mL methanol and filtered with 0.22 pm syringe filter, evaporation and further use for analysis of mycotoxin content.

EXAMPLES

The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

Example 1: Preparation of the sample containing one or more members of mycotoxin analytes, particularly AFB1 and OTA

The certified reference material for AFB1 (BRC 375 < 1 pg/kg) and OTA (wheat RM 471 < 0.6 pg/kg) are obtained from Sigma. Total 146 samples included wheat (35), wheat products (coarsely crushed wheat (20), finely crushed wheat (20), wheat flour (20), refined wheat flour (20), peanut (10), grapes (15) and dog feed (6) obtained randomly from local food stores. Mycotoxin extraction is performed with ground coarsely sample (10 g) and treating with acetonitrile-water (60/40, v/v) (25 mL) by orbital shaking for lh. Extracts are centrifuged at 6500g for 30min and the supernatant was mixed with 15mL chloroform in a separating funnel. The organic phase is collected, dried overnight and solubilised using ImL methanol. The methanol solubilised sample was filtered with 0.22pm syringe filter (Millex-GV), to eliminate the impurities and stored at 4°C for analyses by the POC test strip and HPLC.

For HPLC detection, 1 mL of the extract was concentrated to 50 pL by evaporation at 37°C and the volume was made-up to 1 mL by the acetonitrile: 1 water: acetic acid (99: 99: 2 v/v/v) mobile phase. While detection by developed POC test strip, the methanol solubilized samples are concentrated to 50 pL by evaporation at 37°C and volume is made-up to 1 mL using 0.5X PBS.

Extraction for grapes is carried out by crushing 50 g grapes in mixer grinder. The juice with pulp is mixed with lOmL HaPCU-NaCI solution (33.7 mL, of 85 % orthophosphoric acid and 118 g sodium chloride L'¹ water) and vortexed for 1 min. After addition of 5mL chloroform, the mixture is centrifuged at 2500 g for 15 min to remove the debris and poured into a separating funnel. The clear organic phase at the bottom is transferred to a beaker. The extraction is repeated with 5mL chloroform and the combined extracts are evaporated to dryness under fume hood. The dry extract is dissolved in methanol and filtered through 0.22 pm syringe filter.

Example 2: Determination of mycotoxin, particularly AFB1 and OTA individually or simultaneously by the developed POC test strip and comparison with reference standard detection method i.e. HPLC

The certified reference material (CRM) and 146 food and feed samples for occurrence of AFB1 and OTA are analyzed by the developed POC test strip and compared with the RP-HPLC method. The samples belonging to the category of food and feed are selected for the analysis which includes CRM, whole wheat, crushed wheat, course wheat powder, wheat flour, refined wheat flour, peanut, grapes, dog feed. The mechanism behind the analysis is competitive assay which involves formation of complex between analyte present in sample and the AuNPs- short peptide binding sites and prevention of binding of AuNPs-short peptide with the mycotoxin coated on the T-line. Positive result is indicated as the absence of colour at Test line (Tl, designated for OTA), test-line 2 (T2, designated for AFB1) which shows the presence analyte. Negative result is indicated by the appearance of colour at Tl and/or T2 and control-lines (C). In competitive format of LFA the negative control showed the presence of the three red lines as Tl, T2and C.

For validation of the simultaneous detection of AFB1 and OTA, reverse phase HPLC is carried out by HPLC-LC-20AD (Shimadzu, Japan) using Discovery C18-bonded column using Acetonitrile: Water: Acetic acid (99: 99: 2 v/v/v) as a common mobile phase. Different concentrations of AFB1 and OTA mixtures (50 - 400 ng/mL) are detected in HPLC on the basis of the retention time (RT). AFB1 is detected at the RT of 6.5 min, while OTA is detected at the RT 9.0 min, as depicted in Figure 10A. HPLC method gave a linear response with high correlation R = 0.99 for AFB1 and OTA detection, (Figure 10B, 10C).

The developed multiplexed LFA based on visual readout, the assay interpretation is performed by blinding the samples to the readers. The samples are denoted as ‘true positive’ (TP), ‘true negative’ (TN), ‘false positive’ (FP) or ‘false negative’(FN) based on the comparison of our assay results with those from the HPLC method.

Example 3: Results for AFB 1 and OTA in analyte samples

The CRM of AFB1 shows the presence of 0.41 pg/kg concentration and CRM of OTA showed the presence of 0.05 pg/kg concentration, respectively by HPLC method. The LFA showed presence of Tl, T2 and C indicates low concentration of OTA and AFB1 in the CRM sample by LFA.

Among the 146 samples, 26 wheat and wheat product samples and 4 dog feed samples shows the presence of AFB1 by HPLC. 117 samples tested negative in total for AFB1 and 30 shows the presence of AFB1 (< 1 pg/kg) by HPLC (Table 7). The POC test strip shows negative result in total of 116 samples for AFB1 and 30 samples shows false negative test (Table 3). False positive test is not observed for AFB 1 by the POC test strip.

Out of the 146 samples, 11 wheat and wheat product samples, 2 peanut samples, 1 grape and 6 dog feed samples shows the presence of OTA by HPLC. In total 119 samples were negative for OTA and 20 samples showed the presence of OTA (< 5 pg/kg) as detected by HPLC (Table 7). While, by POC test strip 119 samples tested negative and 27 samples gave false negative test (Table 7).

Table 7: Tabulated results for the AFB1 and OTA analysis in analyte samples

Example 3: Statistical analysis

As the developed multiplexed LFA based on visual readout, the assay interpretation is performed by blinding the samples to the readers. The samples are denoted as ‘true positive’ (TP), ‘true negative’ (TN), ‘false positive’ (FP) or ‘false negative’ (FN) based on the comparison of our assay results with those from the HPLC method.

The detection sensitivity and specificity of the developed LFA are calculated s follows:

Sensitivity (%) = TP/TP+FNxlOO

Specificity (%) = TN/TN+FPxlOO

Positive predicted value (%) = TP/TP+FPxlOO

Negative predicted value (%) = TN/TN+FNxlOO

Assay accuracy (%) =TP+TN/TP+TN+FP+FNxlOO

Cohen's kappa coefficient to agreement (Cohen, 1960) is calculated to compare the two detection methods as follows:

Cohen's kappa=Pr (a) -Pr (e)/l-Pr (e)

Where Pr (a) is the observed relative agreement among raters and Pr (e) is the theoretical probability of chance agreement expected among raters.

There was no false positive test observed by POC test strip for OTA detection. The optimized multiplexed POC test strip showed detection sensitivity and specificity of 53 and 100 % respectively with an assay accuracy of 87.9 % (Table 8). The positive predictive value was 100 % and negative predictive value was 86.3 %. The Cohen's kappa coefficient was 0.70 showing a substantially agreement of the two methods.

The optimized multiplexed assay shows diagnostic sensitivity and specificity of 52 and 100 %, respectively and an assay accuracy of 82.8 % (Table 8). The positive and negative predictive values obtained are 100 and 79.4 %, respectively. The Cohen's kappa coefficient is 0.63 showing a substantially agreement of the two methods.

Table 8A: Statistical parametersforAFB 1 detection

Table 8B: Statistical parameters for OTA detection

ADVANTAGES OF THE INVENTION

• Rapid and on-site detection which is independent of any equipment

• Individual as well as simultaneous multiplexed detection of sample comprising of mycotoxin analytes particularly Aflatoxin Bl and Ochratoxin

• Detection of permissible level mycotoxin as per the cut-off of 175 ng of most standard guidelines (FSSAI AFB1 15 pg/kg and OTA 20 pg/kg)

• Assay time 25-30 min

Claims

Claims We Claim,

1. A POC test strip for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously, comprising; a. A sample site for applying the sample with one or more members of mycotoxin analytes particularly Aflatoxin, Ochratoxin alone or in combination; b. A conjugated pad site, pre -treated and loaded with AuNPs- biotinylated AFB1 peptide and AuNPs-biotinylated OTA peptide (pooled together loaded at one site) c. A nitrocellulose membrane sheet of pore size of 8pm pore and 25mm, loaded with Protein A as a control line, AFB1 and OTA separately as a test line d. An Absorption pad to collect excess waste and to ensure no back flow of the fluid

2. The POC test strips for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously, as claimed in claim 1, wherein the sample is selected from microbially contaminated food products particularly cereals, processed cereals, fruits with high dextrose content, agricultural product particularly wheat, peanut, grapes, animal feed particularly poultry feed, dog feed.

3. The POC test strips for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, as claimed in claim 1, wherein the AuNPs-biotinylated AFB 1 peptide comprises of: a. A short peptide of Seq ID 1 possessing binding affinity for Aflatoxin b. A linker to permit the stereo-chemical binding with toxin and gold nanoparticles for their intended use as detection agents c. A biotin, to enhance the binding of AFB1 specific short peptide (Seq ID 1) to the streptavidin coated gold nanoparticles d. A streptavidin conjugated gold nanoparticles as a detection label The POC test strips for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously, as claimed in claim 1, wherein the AuNPs-biotinylated OTA peptide comprises of : a. A short peptide of Seq ID 2 possessing binding affinity for Ochratoxin b. A linker to permit the stereo-chemical binding with toxin and gold nanoparticles for their intended use as detection agents c. A biotin, to enhance the binding of OTA specific short peptide (Seq ID 2) to the streptavidin coated gold nanoparticles d. A streptavidin conjugated gold nanoparticles as a detection label The POC test strips for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously, as claimed in claim 1, wherein the short peptide of Seq ID 1 present in AuNPs-biotinylated AFB1 peptide is having a peptide sequence of:

N’-(Lys Ser Lys Pro Arg)-C’ The POC test strips for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously, as claimed in claim 1, wherein the short peptide of Seq ID 2 present in AuNPs-biotinylated OTA peptide is having a peptide sequence of:

N’-(Ser Phe Asn His Pro Lys)-C’ The POC test strips for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously, as claimed in claim 1, wherein the linker is a short chain amino acid residues with atleast three noninteracting amino acid, particularly Lysine, Serine and Glycine. A method for developing the POC test strips for simultaneous analysis of a sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin, individually or simultaneously comprising steps of: a. Rationally selecting the short peptide with Seq ID 1 or 2 having affinity for Aflatoxin and Ochratoxin respectively; b. Modifying the selected short peptide by linking with the linker; c. Biotinylating the linker-short peptide conjugate at N terminal of the linker by contacting it with biotin molecule (biotin- linker purchased) d. Rationally selecting the short peptide with Seq ID 1 or 2 having affinity for Aflatoxin and Ochratoxin respectively; e. Selecting the linker to modify the short peptide with Seq ID 1 or 2 depending characteristics such as Hydrogen bonding, hydrophobicity, solubility f. Conjugating the biotin tagged linker modified short peptide (30pL, lug/lOOul PBS, pH 7.0) with gold nanoparticles coated with streptavidin (500pL) g. Loading pre-treated conjugated pads with AuNPs-biotinylated AFB1 peptide and AuNPs-biotinylated OTA peptide (0.8OD) and drying it at 30-40°C for l-3h at a band cut of 4mm h. Coating of AflatoxinBl (10 mg/ml, 4 times) and Ochratoxin A (10 mg/ml , 4 times) as test linel and test line 2 respectively and printing of Protein A (5 mg/ml in PBS) as control line on the nitrocellulose membrane sheet (8pm) i. Passing sample diluted with Phosphate buffer solution (pH 7) containing one or more mycotoxin analyte particularly AFB 1 and OTA A method for the analysis of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin by the POC test strips, comprising the steps of: a. Preparing the sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin by diluting with Phosphate buffer solution (0.5x) b. Adding the prepared sample containing one or more members of mycotoxin analytes, particularly Aflatoxin and Ochratoxin to the sample site (100 pl) c. Passing of the sample to the conjugated pad site and nitrocellulose membrane sheet and absorption pad d. Visualizing the color change from white to dark pink this at test line 1, test line 2, control line due to reaction of AuNPs- biotinylated AFB1 peptide and AuNPs-biotinylated OTA peptide with AFB1 and OTA and protein A present the test sample respectively The method for the analysis of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin by the POC test strips, as claimed in claim 7, wherein the permissible level of analysis for mycotoxin is 175ng/ml or less. The method for the analysis of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin by the POC test strips, as claimed in claim 7, wherein the analysis time is 25-30min. A kit for detection of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin, individually or simultaneously, wherein the kit comprising; a. At least one POC test strips; b. a sealable plastic tube containing a phosphate buffer saline (0.5X) c. a dropper characterized to aspire the prepared sample The kit for detection of a sample containing one or more members of mycotoxin analytes particularly Aflatoxin and Ochratoxin, individually or simultaneously as claimed in claim 12, wherein a. Limit of detection of the kit is 175ng/ml b. Detection time is 25-30min c. Detection by appearance by colored line