Patent Application
20230015484
The present application relates to detection of food allergens. In particular, the present application relates to an electrochemiluminescence immunosensor for the detection of trace amount of tropomyosin, and a method thereof. The present invention also relates to a nanocomposite for the detection of the tropomyosin.
Food allergy is defined by an abnormal immune response after eating a particular food, and is one of the global health concerns for both children and adults. Ingestion of such allergens may instigate mild and acute symptoms such as diarrhea, nausea, and anaphylaxis. One of the most prevalent food allergens present in marine food diet such as shrimps, shellfish, crabs, oyster, squid, and other invertebrates is tropomyosin. Tropomyosin is a two-stranded alpha-helical, thin filament protein found in cytoskeletons and the tropomyosin found in marine food diet is the heat-stable food allergens. As the allergenicity due to such allergen varies in different individuals, there is no immunosensor developed yet to detect such allergen. Furthermore, even a trace amount of such allergen can be harmful if consumed by those allergic to it. Hence, it is very important to monitor such allergens.
There are many point-of-care (POC) biosensors available which can monitor food allergens. The biosensors are defined as analytical devices which are found to be highly sensitive in detecting foodborne-pathogens and allergens. Such biosensors can similarly detect food allergens. There are different types of biosensors depending on the requirements, for example enzymatic, DNA-based, and immunosensors. There are many conventional methods for the detection of food allergens such as radioallergosorbent test (RAST), enzyme allergosorbent test (EAST), rocket immunoelectrophoresis (RIE), enzyme-linked immunosorbent assay (ELISA), dot immunoblotting, protein chip, etc. However, as biosensors have high selectivity and rapid in obtaining results, they are preferred over the conventional methods. There are some biosensors developed for the detection of tropomyosin, for example electrochemical immunosensor, fluorescent aptasensor, etc. The electrochemical immunosensor includes magnetic beads functionalized with carboxyl groups and customized magnetic nanoparticles on a screen-printed carbon electrode. The fluorescent aptasensor includes magnetic aptamer-immobilized detection probe. However, these strategies have a very low sensitivity of detecting tropomyosin.
Therefore, there exists a need for developing biosensor or immunosensor that have higher sensitivity of detecting even trace amounts of tropomyosin.
In a first aspect, the present application discloses a nanocomposite film. The film includes carbon nanohorns (CNHs—OH); Nafion® perfluorinated resin solution; and magnetic nanoparticles. The magnetic nanoparticles are iron oxide supported by palladium-based nanoparticles. The film further includes at least 0.1 mg/mL of the carbon nanohorns; and at least 0.1% of each of the Nafion® perfluorinated resin solution, and magnetic nanoparticles. The carbon nanohorns are dispersed in the Nafion® perfluorinated resin solution, thereby getting oxidized. An antibody may be entrapped on the film via electrostatic interaction and physical adsorption.
In a second aspect, the present application discloses an electrochemiluminescence immunosensor. The immunosensor includes an electrode functionalized by a nanocomposite film. The film further includes carbon nanohorns dispersed in Nafion® perfluorinated resin solution. The polymeric solution is further stabilized by magnetic nanoparticles. The immunosensor includes at least 0.1 mg/mL of the oxidized carbon nanohorns; and at least 0.1% of iron oxide-palladium nanoparticles being immobilized on the SPE. In some embodiments, the immunosensor further includes measuring electrical signal through a [Ru(bpy)3]2+/TPrA electrochemiluminescence system. The system has [Ru(bpy)3]2+ as a luminophore and Tripropylamine (TPrA) as a co-reactant on an interface between the nanocomposite film and the modified electrode. A redox reaction of electron transfer takes place between the modified electrode's surface and [Ru(bpy)3]2+/TPrA ECL system.
The immunosensor is a point-of-care (POC)-based device. The immunosensor is configured to work in the range of 100 ng/mL to 1 fg/mL, and has tendency to detect even traces amount of the tropomyosin. The immunosensor is capable of detecting traces even less than 1 fg/mL, hence having high specificity for Tro-Ag detection in food products with distinguished repeatability.
In yet another aspect, the present application discloses a method for detecting an analyte in a food sample. The method involves fabricating an immunosensor. The fabrication further involves a number of steps, sequence thereof may be exemplary for the skilled persons to understand the present application. The fabrication firstly includes preparing at least 0.1 mg/mL of an oxidized solution of carbon nanohorns. The oxidized solution can be prepared by dispersing the carbon nanohorns in at least 0.1% of Nafion® perfluorinated resin solution. The fabrication involves synthesizing magnetic nanoparticles simultaneously through another method involving a number of steps sequence thereof again may be exemplary for the skilled persons to understand the present application. The method involves initial mixing of at least 4 mL of ultrapure water and at least 10 mM of ascorbic acid, followed by adding at least 10 mM of K2PdCl6 thereto. At least 4 mL of 0.1% of magnetic nanoparticles such as Fe3O4 are dispersed in ultrapure water. The method further includes stirring the above solution at 700 rpm for at least 1 hour at a temperature of 60° C. Thereafter, magnetic separation is performed for at least 3 minutes and washing thereof with ultrapure water, preparing the magnetic iron oxide-palladium nanoparticles. The method includes further redispersing the magnetic nanoparticles in at least 2 mL of the ultrapure water.
Finally, the fabrication method involves combining the oxidized solution of carbon nanohorns with the iron oxide-palladium nanoparticles, followed by stirring for at least 3 hours at 60° C., synthesizing a nanocomposite film. The method involves dropping at least 34 of the synthesized nanocomposite film onto a screen-printed electrode until completely drying, fabricating the immunosensor. Thereafter, at least 34 of the food sample is loaded onto the immunosensor, followed by incubating for at least 30 minutes, forming an immunocomplex between a binding agent on the immunosensor and the sample. The electrode undergoes washing with at least 10 m-M of Phosphate-buffered saline (PBS) buffer at pH 7.4, removing unreacted proteins from the sample. An electrical signal may be detected on the electrode, thereby detecting the analyte concentration. In some embodiments, the analyte is a tropomyosin, and the binding agent is an antibody. The electrode is a carbon screen-printed electrode.
The accompanying figures (FIGs.) illustrate embodiments and serve to explain principles of the disclosed embodiments. It is to be understood, however, that these figures are presented for purposes of illustration only, and not for defining limits of relevant applications
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the exemplary embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Any alterations and further modifications of the inventive features illustrated herein, and any additional applications of the principles of the invention as illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention.
The present application discloses a nanocomposite film which can be utilized as a carrier of antibodies to bind with Tropomyosin in a food sample. Tropomyosin is a heat stable antigen found in food and is considered an allergen. It can be found in marine staple diet such as shrimp, oyster, crabs, shell fish, and other invertebrates. If tropomyosin is consumed by children and/or adults that are allergic to it thereto can cause diarrhea, nausea, etc. The film includes carbon nanohorns (CNHs-OH); Nafion® perfluorinated resin solution; and magnetic nanoparticles. The magnetic nanoparticles are iron oxide supported by palladium-based nanoparticles. The film further includes at least 0.1 mg/mL of the carbon nanohorns; and at least 0.1% of each of the Nafion® perfluorinated resin solution, and magnetic nanoparticles. The carbon nanohorns are dispersed in the Nafion® perfluorinated resin solution, thereby getting oxidized. An antibody may be entrapped on the film via electrostatic interaction and physical adsorption.
In a second aspect, the present application discloses an electrochemiluminescence immunosensor. The immunosensor includes an electrode functionalized by the nanocomposite film. The immunosensor includes at least 0.1 mg/mL of the oxidized carbon nanohorns; and at least 0.1% of iron oxide-palladium nanoparticles being immobilized on the electrode. The electrode is a carbon-printed screen electrode. In some embodiments, the immunosensor further includes measuring electrical signal through a [Ru(bpy)3]2+/TPrA electrochemiluminescence system. The system has [Ru(bpy)3]2+ as a luminophore and Tripropylamine (TPrA) as a co-reactant on an interface between the nanocomposite film and the modified electrode. A redox reaction of electron transfer takes place between the modified electrode's surface and [Ru(bpy)3]2+/TPrA ECL system.
The immunosensor is a Point-of-care (POC)-based device. The immunosensor is configured to work in the range of 100 ng/mL to 1 fg/mL, and has tendency to detect even traces of the tropomyosin. The immunosensor is capable to detect traces even less than 1 fg/mL, hence having high specificity for Tro-Ag detection in food products with distinguished repeatability.
Rabbit polyclonal anti-tropomyosin (anti-Tro) and natural tropomyosin purified from Carolina shrimp (Tro-Ag) were obtained from Indoor Biotechnologies, Inc. (Bangalore, India). Both anti-Tro and Tro-Ag were further diluted in a 10-mM PBS (pH 7.4) and stored at −20° C. Iron oxide water dispersion (Fe3O4), 99.5+%, 15-20 nm, and 20% weight in H2O, was obtained from US Research Nanomaterials, Inc. (TX, USA). A 10-nm Life Science Gold Colloid was purchased from the BBI™ Solutions (Crumlin, UK). Bovine serum albumin (BSA, 96-99%), L-ascorbic acid (AA, ACS reagent, 99%), magnesium chloride hexahydrate (MgCl2.6H2O, ACS reagent, 99.0-102.0%), Nafion® perfluorinated resin solution, oxidized carbon nanohorns (CNHs-OH), potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), potassium ferrocyanide, potassium ferricyanide, potassium hexachloropalladate(IV) (K2PdCl6, 99%), sodium azide (NaN3), sodium chloride (NaCl), sodium phosphate dibasic (Na2HPO4, for molecular biology, 98.0%), tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]Cl2.6H2O), tripropylamine (TPrA) with 98% purity, and tris hydrochloride (Tris-HCl) were bought from Sigma-Aldrich Co. (Saint Louis, USA). A 10-mM phosphate-buffered saline (PBS) of pH 7.4 was prepared by dissolving KCl, NaCl, KH2PO4, and Na2HPO4 in double distilled water. [Ru(bpy)3]Cl2.6H2O and TPrA were also dissolved in double distilled water. Meanwhile, binding buffer (pH 7.2) was prepared by mixing 10-mM Tris-HCl, 150-mM NaCl, 10-mM KCl, and 2.5-mM MgCl and made up to the desired volume with double distilled water. It is contemplated that Nafion® referred hereinafter relates to Nafion® perfluorinated resin solution and has been purchased for experimental purposes.
Preparation of the Nanocomposite film Firstly, a 0.1 mg/mL of oxidized solution of carbon nanohorns (CNHs-OH) is prepared. Such a solution was prepared by dispersing the carbon nanohorns in 0.1% Nafion® and the mixture was sonicated for 1 hour. Simultaneously, the Fe3O4@Pd core-shell nanoparticles are synthesized. The synthesis involved initial mixture of 4-mL ultrapure H2O and 200-μL ascorbic acid [10 mM], followed by adding 200-μL K2PdCl6 [10 mM]. Thereafter, 4 mL of 0.1% of Fe3O4 is dispersed in ultrapure H2O and stirred at 700 rpm for 1 h (60° C.). The nanoparticles further underwent magnetic separation for 3 min and underwent washing with ultrapure water three times. The nanoparticles were redispersed in 2 mL of ultrapure H2O. Thereafter, at least 0.1 mg/mL of CNHs-OH in 0.1% Nafion® was combined with the Fe3O4@Pd nanoparticles, in a 1:1 volume ratio in separate glass vials. The mixture (nanocomposite film) was then stirred for 3 h at 60° C. and stored at 4° C. for further use.
Fabrication of Immunosensor
The immunosensor was fabricated by firstly dropping 3 μL of the synthesized the nanocomposite film onto the carbon working electrode of the SPE until it is completely dried. Then, 3 μL of 10 pg/mL anti-Tro was spiked onto the modified working electrode and incubated for 30 min to allow it to be entrapped by the Nafion® film. Afterwards, the electrode's surface was washed with 10-mM PBS (pH 7.4) to remove unbound anti-Tro. Next, 3 μL of 1% BSA dissolved in 0.1% NaN3 was drop-casted onto the working electrode and left for 45 min. The same buffer (10-mM PBS), pH 7.4 was used for the final washing step, thus completing the preparation of the immunosensor. All of the fabrication processes were performed at room temperature (20° C.±1° C.), in a desiccator. The fabricated immunosensor was then stored at 4° C. until needed. The graphical illustration of the modification on the working electrode of the carbon SPE is shown in
Electrochemiluminescence (ECL) response of the samples was inspected using BDTeCLP100—an ECL signal recorder, purchased from BioDevice Technology Ltd. (Kanazawa, Japan). The photon counting time was set to 500 m sec, the measurement point was set to 60, a scan rate of 50 mV/s was used, and the potential range was set from 0 to 1.0 V. Disposable screen-printed electrodes (SPE) were acquired from BioDevice Technology Ltd. (Kanazawa, Japan), which consisted of carbon working electrode (with working diameter of 2.64 mm2), counter electrode, and silver reference electrode. Preceding the analyses, 3 μL of the sample was dropped onto the fabricated biosensor and incubated for 30 min, to allow the formation of immunocomplex (between Ab-sample). Subsequently, the electrode was washed with 10-mM PBS buffer, pH 7.4, to remove the unreacted proteins in the sample. ECL measurements were carried out at room temperature (20° C.±1° C.) with 800 μM [Ru(bpy)3]2+ and 20 mM TPrA, mixed in 10 mM of PBS buffer (pH 7.4). All of the measurements recorded were obtained at a working potential of 1.0 V.
All of the electrochemical analyses were performed with Autolab PGSTAT101 III (Metrohm, Netherlands) combined with its accompanying software, Nova 1.10. Identically modified SPE chips were utilized as the platform for the electrochemical-based detections. All analyses were performed at room temperature of 20° C.±1° C., and a 5-mM [Fe(CN)6]3−/[Fe(CN)6]4− prepared in 10-mM PBS, pH 7.4, was used as the redox mediator for electrochemical studies. Each of the analyses was replicated three times.
Imitation crab stick, oyster sauce, and rice crackers were purchased from a local store. The food extracts were then prepared according to the manual accompanying the Allergen Extraction kit purchased from Neogen® (USA), with slight modification, Firstly, the extraction solution that consisted of 10-mM PBS of pH 7.4 (included in the kit) was prepared. The extraction solution was pre-heated by immersing the bottle containing the solution in a water bath at 60° C. A 5 g of finely crushed/chopped samples was weighed or 5 mL of liquid sample was placed into a 250-mL bottle. One scoop of extraction powder (included in the kit) was added into the bottle, followed by 125 mL of the pre-heated extraction buffer. The bottle was then sealed tightly to avoid splashing during the extraction process. The bottle was then left shaking at 200 rpm in a water bath at 60° C. for 30 min, after which the bottle was taken out of the water bath and left to stand for 10 min. Finally, 1 mL of the supernatant was pipetted into a fresh, clean microcentrifuge tube and cooled to room temperature prior to analysis. Three microliters of these samples were used for their individual analysis via ECL technique.
Apart from ECL analyses, cyclic voltammetry (CV) (
The CV graph after each step of fabrication relied on the redox (reduction-oxidation) reaction of [Fe(CN)6]3−/[Fe(CN)6]4−. The peak current was markedly reduced as the nanocomposite became completely immobilized onto the surface of the working electrode. Such an immobility may be due to the presence of negatively charged sulfonic acid groups of the Nafion® perfluorinated resin solution, causing a repulsion between the redox species and the electrode's surface. A significant decrease in peak current was observed as the peak current shifted towards the positive potential denoting successful addition of anti-Tro and BSA, which are both negatively charged at pH 7.4, creating stearic hindered environment for rapid electron transfer to take place. After incubation with Tro-Ag, the current decreased further as the Ab-Ag immunocomplexes were formed.
As shown in
Various ECL luminophore were analyzed for the purpose of finding the optimal luminophore to be used further studies. 100 μM luminol, 1 mg/mL CdTe QDs (cadmium telluride quantum dots), and 800 μM tris(2,2′-bipyridyl)ruthenium(II) ([Ru(bpy)3]2+) were investigated as shown in
The ECL analyses was progressively conducted, starting from the bare carbon SPE, upon addition of nanocomposite, after the immobilization of blocking agent (0 pg/mL Tro-Ag), and finally in the presence of 100 pg/mL Tro-Ag. Immobilization of the CNHs-OH/Nafion/Fe3O4@Pd nanocomposite was deduced to be successful as the ECL intensity was improved by ˜1.5 times as shown in
The obtained Nyquist plots (
Field emission scanning electron microscopy (FE-SEM) was additionally performed with bare SPE and modified SPEs with different constituents and combinations of the nanocomposite as shown in
The efficacy of the fabricated ECL immunosensor as a quantitative assay was investigated by varying the concentration of the target antigen (Tro-Ag) from 100 ng/mL to 1 fg/mL. These concentrations were selected in order to focus on developing the immunosensor that is able to detect trace levels of tropomyosin. The resulting data were plotted into two separate graphs. As the concentration of Tro-Ag increased from 1 fg/mL to 10 pg/mL (
The selectivity of our bioassay was further validated using raw and processed food samples, which typically constituted of numerous proteins and other components that might interfere with the signal production and therefore result in false positive or false-negative results. For this purpose, five different allergen proteins (antigens) at concentration of 100 pg/mL were selected, spiked, and incubated under optimal conditions onto the developed immunosensor. The selected allergens included tropomyosin (Tro), bovine serum albumin (BSA), casein, lysozyme (Lyso), and ovalbumin (OVA) as they are some of the common constituents of processed food products. The observed ECL signals were recorded and expressed as a bar graph in
Following this, the repeatability of this developed immunosensor was investigated by incubating 100 pg/mL Tro-Ag with five stand-alone fabricated electrodes (
The present application discloses a method 1400 for detecting analyte in a food sample. The method 1400 involves a number of steps, sequence thereof may be exemplary to understand the skilled persons in the art. The method 1400 involves preparing at least 0.1 mg/mL of an oxidized solution of carbon nanohorns by dispersing the carbon nanohorns in at least 0.1% of Nafion® perfluorinated resin solution at step 1402. Thereafter, the method 1400 involves synthesizing magnetic nanoparticles simultaneously at step 1404. The method 1400 involves combining the oxidized solution of carbon nanohorns with the iron oxide-palladium nanoparticles, followed by stirring for at least 3 hours at 60° C., synthesizing a nanocomposite film at step 1406. Thereafter, the method 1400 involves dropping at least 3 μL of the synthesized nanocomposite film onto a screen-printed electrode until completely drying, fabricating the immunosensor at step 1408. At step 1410, at least 3 μL of the food sample is loaded onto the immunosensor, followed by incubating for at least 30 minutes, forming an immunocomplex between a binding agent on the immunosensor and the sample. The electrode undergoes washing with at least 10 m-M of Phosphate-buffered saline (PBS) buffer at pH 7.4, removing unreacted proteins from the sample at step 1412. The method 1400 involves monitoring an electrical signal developed on the electrode at step 1414, followed by finally detecting the analyte/antigen (Tropomyosin) at step 1416.
As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means+/−5% of the stated value, more typically +/−4% of the stated value, more typically +/−3% of the stated value, more typically, +/−2% of the stated value, even more typically +/−1% of the stated value, and even more typically +/−0.5% of the stated value.
Throughout this disclosure, certain embodiments may be disclosed in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
In the application, unless specified otherwise, the terms “comprising”, “comprise”, and grammatical variants thereof, intended to represent “open” or “inclusive” language such that they include recited elements but also permit inclusion of additional, non-explicitly recited elements.
It will be apparent that various other modifications and adaptations of the application will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the application and it is intended that all such modifications and adaptations come within the scope of the appended claims.
