Patent Application
20140248629
1. Technical field
The embodiments herein generally relate to the field of biosensors and particularly to the nano-biosensors used for diagnosing diseases. The embodiments herein more particularly relate to a method of detecting malarial parasites by using a dipstick type nano-biosensor. The embodiments herein further relate to a method of detecting Plasmodium vivax and Plasmodium falciparum.
2. Description of the Related Art
Arthropod-borne infections, such as malaria which are transmitted by mosquitoes, are often quite serious resulting in a significant morbidity and even death. The figures released by the World Health Organization in the World Health Report -1996 shows that 2.1 million deaths were caused by malaria alone in 1995 (Day, “Scourge of infections Kills Third World's Young” in New Scientist, 150(2031): p6, 1996).
Malaria is a mosquito borne infectious disease of humans and other animals and is caused by protists of the genus Plasmodium. A protozoan of the genus Plasmodium, which includes P. falciparum, P. vivax, P. ovate and P. malariae, causes malaria in humans. P. falciparum, which can result in a potentially fatal infection, is widespread throughout the tropics and therefore constitutes an important health threat/hazard for millions of people. P. vivax is also widespread and because of its propensity for successive relapse from liver and consequent toxicity, it constitutes an important cause of morbidity in tropical regions. P. ovale and P. malariae are less common and both of them cause low-grade, chronic diseases and the latter infection often causes a disruption of kidney function through immune-complex deposition.
These protists first infect liver and then act as parasites within Red Blood Cells (RBC) causing symptoms that typically include fever and headache in severe cases progressing to coma or death.
Accurate methods for detecting malaria and other arthropod-borne infections are necessary to identify the infected individuals so as to properly direct a therapy and for identifying further sources that may increase a spread of diseases. The standard and most cost-effective method for detecting the malaria pathogens in mosquitoes involves an isolation of sporozoites from the salivary glands and an enumeration using a phase microscopy on a haemocytometer. However, the method is not very specific and is very much labor intensive. This method requires an effective microscope and skilled technicians. This method is not generally feasible in many regions of the tropics.
The malaria is typically diagnosed by a microscopic examination of blood using blood films. The malaria is also diagnosed by using the antigen based Rapid Diagnostic Tests (RDTs) such as dipsticks and Polymerase Chain Reaction (PCR) based tests.
The microscopic examination of a blood smear reveals the presence of the malarial parasites inside the blood cells. The blood is the most frequently used sample for diagnosis as an invasive specimen while the saliva and the urine have also been investigated as an alternative forming non invasive specimens. But experienced microbiologists and skilled persons are required for these methods. Although the polymerase chain reaction (PCR) based tests have been developed, these are not widely implemented in malarial endemic regions due to their complexity.
The dipsticks are a new generation of antigen based rapid diagnostics tests which are sensitive and the dip sticks are a reliable device for the diagnosis of the malaria. Initially the glutamate dehydrogenase of the P. falciparum was used as an antigen. It is now replaced by the lactate dehydrogenase. The nucleic acid sequence based application (NASBA) products (RNA nucleotides) are used along with antibodies that are labeled with the digoxigenin-uridine triphosphate (dig-UTP). The labeled NASBA products are coated on the nanoparticles. The dipstick based tests allow a rapid diagnosis of the malaria by people who are not otherwise skilled in the laboratory techniques for diagnosing the malarial parasites. Also the dipsticks find their application in field diagnosis where there are no diagnosis centers. None of the rapid tests are currently as sensitive as thick blood film, or cheap. A major drawback in the use of all current dipstick methods is that the result is qualitative.
The U.S. Pat. No. 7,427,483 discloses PCR reaction for amplification and detection of Plasmodium falciparum DNA (a highly conserved region of Plasmodium falciparum MSP1 gene) and final detection is done by Enzyme Linked Immunosorbent Assay (ELISA) procedure. Further the patent discloses the use of polymerase chain reaction (PCR) to label products. The method is specific for C-terminal region of MSP 1 gene. Because of the method and protocol of ELISA, this method requires more time for delivering the results and hence a diagnosis will be delayed. Such a method cannot cope with a great number of infected persons in the area where malaria is prevalent and the number of inspection experts is insufficient.
Another prior art by the U.S. patent No. 2011/0171749A1 discloses the biosensor based on antigen-antibody reaction. Further the biosensor is mainly designed for detecting the Escherichia coli and utilizes polymer coated magnetic nanoparticles. This method is specific to bacterial detection and not for malarial parasite detection. Further this method requires magnetic field which makes the diagnosis complex.
Yet another prior art by U.S. Pat. No. 5,827,681 discloses light microscope for detecting opaque hemozoin particles and has used multiple champers. Hence this method requires microbiological skills. Further the method discloses a use of microscope which limits the use of the invention in laboratory and not in the field conditions. Such a method cannot cope with a great number of infected persons in the area where malaria is prevalent and the number of inspection experts is insufficient.
Yet another prior art by U.S. Pat. No. 5,792,609 discloses a use of a primer for detecting the malarial parasites which is not labeled and hence the method makes it difficult to detect the binding of template sequence and the primer. Hence a monitoring of the primer and template sequence binding to provide a desired result is not assured.
According to yet another prior art, the immune sensor is based on the antigen-antibody reaction. The immune sensor detects the HRP-2 of the Plasmodium falciparum in blood. It uses the disposable screen printed electrodes (SPE's) modified with multiwall carbon nanotubes and the gold nanoparticles. This makes the invention complex as the electrodes are used. Further the article discloses that the final results are obtained based on the microscopic results. Hence the biosensor cannot be used in the field conditions or the places where epidemic of malaria is prevailing.
Hence, there is a need to develop a device which is simple, efficient and sensitive, and which does not require a use of any other instrument while handling. Also, there is need to develop a device that does not require microbiological skills especially in places where there are no diagnostic centers. Further simple assays are much needed in the art to improve the availability of malarial diagnosis. Also there is a need to provide better treatment options to those in need, and to differentiate between symptomatic and asymptomatic disease.
The above mentioned shortcomings, disadvantages and problems are addressed herein and which will be understood by reading and studying the following specification.
The primary object of the embodiments herein is to provide a dipstick nano-biosensor with the gold nanoparticles conjugated to an antibody for diagnosis of the malarial parasites.
Another object of the embodiments herein is to provide a dipstick nano-biosensor for diagnosis of the Plasmodium falciparum and Plasmodium vivax.
Yet another object of the embodiment herein is to provide dipstick nano-biosensor with 90% sensitivity for detecting the Plasmodium falciparum and Plasmodium vivax.
Yet another object of the embodiments herein is to provide sensitive and reliable method for diagnosing malaria in the field conditions, where the diagnostic centers are not present.
Yet another object of the embodiment herein is to provide a potable and a sensitive dipstick nano-biosensor device.
Yet another object of the embodiment herein is to combine a molecular method of NASBA (Nucleic Acid Sequence Based Amplification) with the nanotechnology (use of gold nanoparticles) for preparing the dipstick nano-biosensor.
Yet another object of the embodiments herein is to provide a dipstick nano-biosensor based on the amplification of 18 srRNA by NASBA method.
Yet another object of the embodiments herein is to label NASBA products with the Digoxigenin-11-UTP.
Yet another object of the embodiments is to conjugate the labeled NASBA products with anti digoxigenin conjugated-nano gold particles.
Yet another object of the embodiments herein is to provide a dipstick nano-biosensor comprising two control zones for confirming the diagnostic results of the malarial parasites.
Yet another object of the embodiments herein is to provide a dipstick nano-biosensor so that the results are observed after performing oligochromatography and a microscope is not needed.
These and other objects and advantages of the embodiments herein will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings.
The various embodiments herein provide a gold nanoparticle based dipstick nano-biosensor for detecting plasmodium falciparum and plasmodium vivax and a mehtod of synthesizing the same. The embodiments herein provide a new innovative method for a diagnosis of the malarial parasites. The gold nanoparticles based dipstick nano-biosensor is used in a new generation of antigen based rapid and sensitive diagnostic test, and the dipstick nano bio sensor is a reliable device for diagnosing the Plasmodium vivax and Plasmodium falciparum.
According to an embodiment herein, a dipstick nano-biosensor is provided for detecting Plasmodium vivax and Plasmodium falciparum. The dipstick nano-bio sensor comprising a cellulose membrane, a nitrocellulose membrane, a fibreglass, and a plurality of probes. The cellulose membrane, the nitrocellulose membrane and the fiber glass are coated on a backing plastic plate coated with gold nanoparticles conjugated with an anti-body and wherein the anti-body is an anti-digoxigenin.
According to an embodiment herein, the cellulose and the nitrocellulose membrane are coated on the backing plastic plate with an overlap of 1 mm.
According to an embodiment herein, the gold nanoparticles are conjugated to the anti digoxigenin antibody by a physical adsorption for 1.5 hours in a cold room by a continuous mixing of the gold nanoparticles, and wherein a size of the gold nanoparticles is 0.40-25nm.
According to an embodiment herein, the plurality of the probes comprises a streptavidin, a texas red, a biotin and a fluoresceine.
According to an embodiment herein, the backing plastic plate comprises three regions and wherein the three regions comprise a wicking pad, a conjugate pad, and an absorbent pad respectively.
According to an embodiment herein, the conjugate pad comprises a test zone and a control zone, and wherein the test zone comprises a first test line and a second test line for detecting Plasmodium falciparum and Plasmodium vivax respectively, and wherein the control zone comprises a first control zone and a second control line for checking a correctness of NASBA and oligo chromatography respectively.
According to an embodiment herein, the first test line comprises a nitrocellulose membrane coated with a streptavidin conjugated to a biotin and wherein the second test line comprises a nitrocellulose membrane coated with an anti texas red.
According to an embodiment herein, the first control line comprises a nitrocellulose membrane coated with an antifluorescene antibody and wherein the second control line comprises a nitrocellulose membrane coated with an anti anti-sheep.
According to an embodiment herein, the plurality of probes comprises a 18srRNA and wherein the 18srRNA is isolated from Plasmodium falciparum, Plasmodium vivax and a GAPDH, and wherein the GAPDH is isolated from a blood of healthy human individual, and wherein the isolated 18srRNA and the GAPDH are subjected to an agarose gel electrophoresis to extract specific bands with sequences from an agarose gel.
According to an embodiment herein, the 18srRNA is subjected to a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to get a cDNA.
According to an embodiment herein, the isolated sequences from the agarose gel are cloned into a plasmid vector, and wherein the cloned plasmid vectors are induced into an Escherichia coli, and wherein the plasmids are sequenced.
According to an embodiment herein, 18srRNA and GAPDH are subjected to Nucleic Acid Sequence Based Amplification (NASBA), and wherein NASBA amplified 18sRNA and GAPDH are labelled with digoxigenin-11-UTP.
According to an embodiment herein, the backing plastic plate is coated with digoxigenin-11-UTP labelled 18srRNA, streptavidin, anti texas red, biotin and fluorescein by Airjet 3000.
According to an embodiment herein, a method is provided for synthesizing dipstick nano-biosensor for detecting Plasmodium vivax and Plasmodium falciparum. The method comprises culturing Plasmodium falciarum and Plasmodium vivax, extracting RNA from Plasmodium falciparum and Plasmodium vivax culture and a healthy donor, cloning and sequencing extracted RNA using a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to get a cDNA, performing In vitro transcription of extracted RNA, preparing control positive RNA, isolating GAPDH from a blood of healthy human individual, setting and performing NASBA-DIG labelling of both Pasmodium falciparum and Plasmodium vivax and GAPDH, selecting a plurality of membranes for assembling a dipstick, and wherein the plurality of membranes includes a cellulose membrane, a nitrocellulose membrane, and a fiberglass, and conjugating nano gold particles to anti digoxigenin antibody to form a dipstick.
According to one embodiment herein, the first step for the preparation of a dipstick nano-biosensor and the detection of malarial parasites is a preparation of positive control by an amplification of the RNA's by Nucleic Acid Sequence Based Amplification (NASBA). This step further comprises of RNA extraction, subjecting the isolated RNA's to RT-PCR, cloning the RT-PCR products, sequencing the genes and in vitro transcription. The two species of malarial parasites a Plasmodium falciparum and a Plasmodium vivax are cultured in Pasteur Institute of Iran. The 18 srRNA is isolated from Plasmodium vivax, Plasmodium falciparum continuous invitro culture and healthy human blood. Before subjecting the isolated RNA's to Reverse Transcription—Polymerase Chain Reaction (RT-PCR), the specific probes and the primers are designed in such a way that each specific primer pair does not detect the other species. Further a pair of primers for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, which is a house keeping gene, is designed for checking the correctness of NASBA. The GAPDH gene is co-amplified with the other genes and a specific probe for each gene is designed to detect the NASBA product of each gene.
According to one embodiment herein, the RT-PCR is performed with the specific primers for each gene after designing the specific primers and the probes for each gene. The amplified fragments of a cDNA, thus obtained from the RT-PCR, are cloned into a plasmid vector pDrive (Qiagen) having an ampicillin resistance gene. The cloned plasmid vector pDrive is transformed in the Escherichia coli (DH5). The Escherichia coli are cultivated in LB (Luria Bertani) agar media having an ampicillin, a Xgal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) and an IPTG (isopropylthio-β-galactoside). The colonies are screened and white colonies are taken for plasmid extraction. The plasmids are sequenced and an existence of desired inserts in them is verified.
According to one embodiment herein, an in vitro transcription is further performed to synthesize the RNAs with same size. Hence a linear DNA molecule is needed as a pattern of the template sequence. In this process, PCR reaction is performed by the specific primers having a T7 promoter sequence. The PCR products, which are linear DNA molecules having the T7 promoter, are used in an in vitro transcription reaction as patterns or templates. After this step Plasmodium falciparum and GAPDH RNA are synthesized in laboratory by using the T7 RNA polymerase enzyme which generates RNA from patterns having the T7 promoter sequence. The RNAs generated by the invitro transcription reaction are treated by a DNase I for removing the DNA molecules and are then purified by the Qiazol lysis buffer and stored in −70 ° C. for further use in the NASBA as pattern or template.
According to one embodiment herein, the next step for the synthesis of dipstick nano-biosensor is the dig-labeling (Digoxigenin- labeling) of NASBA products. Hence the NASBA reaction is set and Dig-11-UTP is used instead of usual UTP. The reason for labeling the NASBA products (RNA) with the digoxigenin molecules is that the RNA is required for detection by the antidigoxigenin molecules conjugated with nano gold particles on dipstick nano-biosensor. The RNA labeled with the digoxigenin-11-UTP is now known as dig-UTP. The dig-UTP is now used with a plurality of probes for the dipstick nano-biosensor. The plurality of probes comprises a biotin, a streptavidin, an antifluoresin, an anti-texas red and an IgG antibody of Sheep.
According to one embodiment herein, the next step is the preparation of reagents for the dipstick nano-biosensor. This step comprises of generating an anti sheep or an anti digoxigenin antibody of sheep, which is a conjugation of an anti digoxigenin with the 40 nm nano gold particles, verification of existing sheep antibodies by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The probes such as a Texas red, a biotin, a streptavidin are procured.
According to one embodiment herein, the next step is preparation and assembly of a dipstick nano-biosensor. The dipstick nano-biosensor used for detecting malarial parasites of the genus Plasmodium comprises a cellulose, a nitrocellulose and a fiberglass, the gold nanoparticles coated with an anti-digoxigenin, the NASBA products labeled with the dig-11-UTP and the probes. The probes comprise of a streptavidin, a texas red, a biotin and a fluorescein. The cellulose, nitrocellulose and fiberglass are coated on the backing plastic such that they have 1 mm overlap. Further the nano-biosensor dipstick comprises a control region and a test region. The size of the gold nanoparticles is 25 nm. The gold nanoparticles are coated with an anti-digoxigenin to form a ligand on the membrane.
According to one embodiment herein, a suitable membrane is selected for the dipstick nano-biosensor comprising the gold nanoparticles coated with an anti-digoxigenin antibody. The membranes comprise a cellulose, a nitrocellulose and a fiberglass. The membranes are arranged in a manner such that each membrane overlaps the other by 1 mm. The combined mixture of the Dig-UTP labeled RNA and the probes are added to the membrane.
According to one embodiment herein, the dipstick nano-biosensor further comprises a capture reagent which is prepared in optimum or appropriate concentrations. The capture reagents include a streptavidine, an anti fluoresceine which are obtained from Roche and Biolegend companies respectively and the other reagents such as an anti sheep are prepared in laboratory. For preparing the anti sheep the IgG from the sheep blood are purified by PrG column. The verification process for a presence of existing IgG SDS PAGE is performed. Further the rabbits are immunized by the sheep IgG three times (10 μg/ml). The blood from the immunized rabbits is taken and IgG from the rabbit blood is purified by PrG column. The functionality of the anti sheep obtained from the rabbit blood is verified by performing the SDS-PAGE and the western blotting.
According to one embodiment herein, the dipstick is a portable and light device which contain three parts namely a wicking pad, a conjugate pad, and an absorbent pad. These parts are adhered on a backing plastic in a way that they have 1 mm overlap with each other. The nano gold particles of 0.40 nm are conjugated to anti digoxigenin and are loaded on conjugate pad of fiber glass. The streptavidine , anti fluorsceine and anti anti sheep are loaded on the membrane by air jet 3000 in order to form a test line, a first control line and a second control line respectively.
According to one embodiment herein, the utility and application of the dipstick nano-biosensor is done by the visualization of bands by an oligochromatography. For the oligochromatography process, a proper concentration of each probe and various dilutions of the probes are prepared.
The digoxigenin labeled NASBA products for each gene is mixed with their specific probes and after the hybridization is complete for 10 minutes at 25° C. After hybridization, the mixture is loaded on the conjugate pad. The wicking pad is dipped into hybridization buffer after loading the conjugate pad. The red lines at the bottom of dipstick nano-biosensor shows that the dipstick nano-biosensor can detect hybridization product of Plasmodium falciparum 18srRNA (NASBA product+specific probes). The red line at the top of the dipstick shows that the gold nanoparticles conjugated to the anti digoxigenin are moved along the dipstick nao-biosensor and the gold nanoparticles conjugated to anti digoxigenin could react with the anti sheep coated on the second control line.
The dipstick has detected hybridization product of the Plasmodium falciparum 18srRNA (NASBA product+specific probes) and also hybridization product of the GAPDH (NASBA product+specific probes).
These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.
The other objects, features and advantages will occur to those skilled in the art from the following description of the preferred embodiment and the accompanying drawings in which:
IgG's after purification by PrG column before immunizing a rabbit, according to one embodiment herein.
a illustrates a screen shot indicating the designing of the specific primers for Plasmodium vivax, according to one embodiment herein.
b illustrates a screen shot indicating the designing of the specific primers for the Plasmodium falciparum, according to one embodiment herein.
c illustrates a screen shot indicating the designing of the specific primers for the human GAPDH, according to one embodiment herein.
These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.
In the following detailed description, a reference is made to the accompanying drawings that form a part hereof, and in which the specific embodiments that may be practiced is shown by way of illustration. The embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and it is to be understood that the logical, mechanical and other changes may be made without departing from the scope of the embodiments. The following detailed description is therefore not to be taken in a limiting sense.
The various embodiments herein provide a gold nanoparticle based dipstick nano-biosensor for detecting plasmodium falciparum and plasmodium vivax and a mehtod of synthesizing the same. The embodiments herein provide a new innovative method for a diagnosis of the malarial parasites. The gold nanoparticles based dipstick nano-biosensor is used in a new generation of antigen based rapid and sensitive diagnostic test, and the dipstick nano bio sensor is a reliable device for diagnosing the Plasmodium vivax and Plasmodium falciparum.
According to an embodiment herein, a dipstick nano-biosensor is provided for detecting Plasmodium vivax and Plasmodium falciparum. The dipstick nano-bio sensor comprising a cellulose membrane, a nitrocellulose membrane, a fibreglass, and a plurality of probes. The cellulose membrane, the nitrocellulose membrane and the fiber glass are coated on a backing plastic plate coated with gold nanoparticles conjugated with an anti-body and wherein the anti-body is an anti-digoxigenin.
According to an embodiment herein, the cellulose and the nitrocellulose membrane are coated on the backing plastic plate with an overlap of 1 mm.
According to an embodiment herein, the gold nanoparticles are conjugated to the anti digoxigenin antibody by a physical adsorption for 1.5 hours in a cold room by a continuous mixing of the gold nanoparticles, and wherein a size of the gold nanoparticles is 0.40-25 nm.
According to an embodiment herein, the plurality of the probes comprises a streptavidin, a texas red, a biotin and a fluoresceine.
According to an embodiment herein, the backing plastic plate comprises three regions and wherein the three regions comprise a wicking pad, a conjugate pad, and an absorbent pad respectively.
According to an embodiment herein, the conjugate pad comprises a test zone and a control zone, and wherein the test zone comprises a first test line and a second test line for detecting Plasmodium falciparum and Plasmodium vivax respectively, and wherein the control zone comprises a first control zone and a second control line for checking a correctness of NASBA and oligo chromatography respectively.
According to an embodiment herein, the first test line comprises a nitrocellulose membrane coated with a streptavidin conjugated to a biotin and wherein the second test line comprises a nitrocellulose membrane coated with an anti texas red.
According to an embodiment herein, the first control line comprises a nitrocellulose membrane coated with an antifluorescene antibody and wherein the second control line comprises a nitrocellulose membrane coated with an anti anti-sheep.
According to an embodiment herein, the plurality of probes comprises a 18srRNA and wherein the 18srRNA is isolated from Plasmodium falciparum, Plasmodium vivax and a GAPDH, and wherein the GAPDH is isolated from a blood of healthy human individual, and wherein the isolated 18srRNA and the GAPDH are subjected to an agarose gel electrophoresis to extract specific bands with sequences from an agarose gel.
According to an embodiment herein, the 18srRNA is subjected to a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to get a cDNA.
According to an embodiment herein, the isolated sequences from the agarose gel are cloned into a plasmid vector, and wherein the cloned plasmid vectors are induced into an Escherichia coli, and wherein the plasmids are sequenced.
According to an embodiment herein, 18srRNA and GAPDH are subjected to Nucleic Acid Sequence Based Amplification (NASBA), and wherein NASBA amplified 18sRNA and GAPDH are labelled with digoxigenin-11-UTP.
According to an embodiment herein, the backing plastic plate is coated with digoxigenin-11-UTP labelled 18srRNA, streptavidin, anti texas red, biotin and fluorescein by Airjet 3000.
According to an embodiment herein, a method is provided for synthesizing dipstick nano-biosensor for detecting Plasmodium vivax and Plasmodium falciparum. The method comprises culturing Plasmodium falciarum and Plasmodium vivax, extracting RNA from Plasmodium falciparum and Plasmodium vivax culture and a healthy donor, cloning and sequencing extracted RNA using a Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) to get a cDNA, performing In vitro transcription of extracted RNA, preparing control positive RNA, isolating GAPDH from a blood of healthy human individual, setting and performing NASBA-DIG labelling of both Pasmodium falciparum and Plasmodium vivax and GAPDH, selecting a plurality of membranes for assembling a dipstick, and wherein the plurality of membranes includes a cellulose membrane, a nitrocellulose membrane, and a fiberglass, and conjugating nano gold particles to anti digoxigenin antibody to form a dipstick.
According to one embodiment herein, the first step for the preparation of a dipstick nano-biosensor and the detection of malarial parasites is a preparation of positive control by an amplification of the RNA's by Nucleic Acid Sequence Based Amplification (NASBA). This step further comprises of RNA extraction, subjecting the isolated RNA's to RT-PCR, cloning the RT-PCR products, sequencing the genes and in vitro transcription. The two species of malarial parasites a Plasmodium falciparum and a Plasmodium vivax are cultured in Pasteur Institute of Iran. The 18 srRNA is isolated from Plasmodium vivax, Plasmodium falciparum continuous invitro culture and healthy human blood. Before subjecting the isolated RNA's to Reverse Transcription—Polymerase Chain Reaction (RT-PCR), the specific probes and the primers are designed in such a way that each specific primer pair does not detect the other species. Further a pair of primers for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, which is a house keeping gene, is designed for checking the correctness of NASBA. The GAPDH gene is co-amplified with the other genes and a specific probe for each gene is designed to detect the NASBA product of each gene.
According to one embodiment herein, the RT-PCR is performed with the specific primers for each gene after designing the specific primers and the probes for each gene. The amplified fragments of a cDNA, thus obtained from the RT-PCR, are cloned into a plasmid vector pDrive (Qiagen) having an ampicillin resistance gene. The cloned plasmid vector pDrive is transformed in the Escherichia coli (DH5). The Escherichia coli are cultivated in LB (Luria Bertani) agar media having an ampicillin, a Xgal (5-bromo-4-chloro-indolyl-f3-D-galactopyranoside) and an IPTG (isopropylthio-f3-galactoside). The colonies are screened and white colonies are taken for plasmid extraction. The plasmids are sequenced and an existence of desired inserts in them is verified.
According to one embodiment herein, an in vitro transcription is further performed to synthesize the RNAs with same size. Hence a linear DNA molecule is needed as a pattern of the template sequence. In this process, PCR reaction is performed by the specific primers having a T7 promoter sequence. The PCR products, which are linear DNA molecules having the T7 promoter, are used in an in vitro transcription reaction as patterns or templates. After this step Plasmodium falciparum and GAPDH RNA are synthesized in laboratory by using the T7 RNA polymerase enzyme which generates RNA from patterns having the T7 promoter sequence. The RNAs generated by the invitro transcription reaction are treated by a DNase I for removing the DNA molecules and are then purified by the Qiazol lysis buffer and stored in −70 ° C. for further use in the NASBA as pattern or template.
According to one embodiment herein, the next step for the synthesis of dipstick nano-biosensor is the dig-labeling (Digoxigenin-labeling) of NASBA products. Hence the NASBA reaction is set and Dig-11-UTP is used instead of usual UTP. The reason for labeling the NASBA products (RNA) with the digoxigenin molecules is that the RNA is required for detection by the antidigoxigenin molecules conjugated with nano gold particles on dipstick nano-biosensor. The RNA labeled with the digoxigenin-11-UTP is now known as dig-UTP. The dig-UTP is now used with a plurality of probes for the dipstick nano-biosensor. The plurality of probes comprises a biotin, a streptavidin, an antifluoresin, an anti-texas red and an IgG antibody of Sheep.
According to one embodiment herein, the next step is the preparation of reagents for the dipstick nano-biosensor. This step comprises of generating an anti sheep or an anti digoxigenin antibody of sheep, which is a conjugation of an anti digoxigenin with the 40 nm nano gold particles, verification of existing sheep antibodies by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The probes such as a Texas red, a biotin, a streptavidin are procured.
According to one embodiment herein, the next step is preparation and assembly of a dipstick nano-biosensor. The dipstick nano-biosensor used for detecting malarial parasites of the genus Plasmodium comprises a cellulose, a nitrocellulose and a fiberglass, the gold nanoparticles coated with an anti-digoxigenin, the NASBA products labeled with the dig-11-UTP and the probes. The probes comprise of a streptavidin, a texas red, a biotin and a fluorescein. The cellulose, nitrocellulose and fiberglass are coated on the backing plastic such that they have 1 mm overlap. Further the nano-biosensor dipstick comprises a control region and a test region. The size of the gold nanoparticles is 25 nm. The gold nanoparticles are coated with an anti-digoxigenin to form a ligand on the membrane.
According to one embodiment herein, a suitable membrane is selected for the dipstick nano-biosensor comprising the gold nanoparticles coated with an anti-digoxigenin antibody. The membranes comprise a cellulose, a nitrocellulose and a fiberglass. The membranes are arranged in a manner such that each membrane overlaps the other by 1 mm. The combined mixture of the Dig-UTP labeled RNA and the probes are added to the membrane.
According to one embodiment herein, the dipstick nano-biosensor further comprises a capture reagent which is prepared in optimum or appropriate concentrations. The capture reagents include a streptavidine, an anti fluoresceine which are obtained from Roche and Biolegend companies respectively and the other reagents such as an anti sheep are prepared in laboratory. For preparing the anti sheep the IgG from the sheep blood are purified by PrG column. The verification process for a presence of existing IgG SDS PAGE is performed. Further the rabbits are immunized by the sheep IgG three times (10 μg/ml). The blood from the immunized rabbits is taken and IgG from the rabbit blood is purified by PrG column. The functionality of the anti sheep obtained from the rabbit blood is verified by performing the SDS-PAGE and the western blotting.
According to one embodiment herein, the dipstick is a portable and light device which contain three parts namely a wicking pad, a conjugate pad, and an absorbent pad. These parts are adhered on a backing plastic in a way that they have 1 mm overlap with each other. The nano gold particles of 0.40 nm are conjugated to anti digoxigenin and are loaded on conjugate pad of fiber glass. The streptavidine , anti fluorsceine and anti anti sheep are loaded on the membrane by air jet 3000 in order to form a test line, a first control line and a second control line respectively.
According to one embodiment herein, the utility and application of the dipstick nano-biosensor is done by the visualization of bands by an oligochromatography. For the oligochromatography process, a proper concentration of each probe and various dilutions of the probes are prepared. The digoxigenin labeled NASBA products for each gene is mixed with their specific probes and after the hybridization is complete for 10 minutes at 25° C. After hybridization, the mixture is loaded on the conjugate pad. The wicking pad is dipped into hybridization buffer after loading the conjugate pad. The red lines at the bottom of dipstick nano-biosensor shows that the dipstick nano-biosensor can detect hybridization product of Plasmodium falciparum 18srRNA (NASBA product+specific probes). The red line at the top of the dipstick shows that the gold nanoparticles conjugated to the anti digoxigenin are moved along the dipstick nao-biosensor and the gold nanoparticles conjugated to anti digoxigenin could react with the anti sheep coated on the second control line. The dipstick has detected hybridization product of the Plasmodium falciparum 18srRNA (NASBA product+specific probes) and also hybridization product of the GAPDH (NASBA product+specific probes).
The primers used are:
Plasmodium vivax 18srRNA reverses primer
Plasmodium vivax 18srRNA forward primer
Plasmodium falciparum 18srRNA forward primer
Plasmodium falciparum 18srRNA reverse primer
Plasmodium falciparum 18srRNA probe:
The anti anti-sheep was prepared in laboratory. The anti anti-sheep was then conjugated to nano gold particles and used in dipstick nano-biosensor assembly. The blood from a sheep is extracted (1301). The IgG's (Immunoglobin G) are isolated from the sheep blood by a PrG column to obtaing an isolate (1302).The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is performed for verifying the presence of IgG in the isolate (1303). After SDS-PAGE, a 3 kg rabbit is selected and immunized with sheep IgG, for 3 times at a concentration of 10 μg/μl (1304). After the rabbit is immunized, the blood is extracted from the immunized rabbit (1305). The immunized rabbit produces antibody against the sheep IgG's and the antibodies are called as anti anti-sheep IgG. The IgG's from rabbit blood are purified by PrG column (1306). The purified IgG's from the rabbit blood are subjected to SDS-PAGE and Western blotting for verifying functionality (1307).
a illustrates a screen shot indicating a designing of the specific primers for the Plasmodium vivax, according to one embodiment herein. The primer designed detects specific sequences of the Plasmodium vivax in in vitro transcription.
b illustrates a screen shot indicating a designing of the specific primers for Plasmodium vivax, according to one embodiment herein. The primer designed detects specific sequences of Plasmodium falciparum in in vitro transcription.
c illustrates a screen shot indicating a designing of the specific primers for human GAPDH, according to one embodiment herein. The primer designed detects specific sequences of GAPDH in invitro transcription.
The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments.
It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.
Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the appended claims.
Although the embodiments herein are described with various specific embodiments, it will be obvious for a person skilled in the art to practice the invention with modifications. However, all such modifications are deemed to be within the scope of the claims.
It is also to be understood that the following claims are intended to cover all of the generic and specific features of the embodiments described herein and all the statements of the scope of the embodiments which as a matter of language might be said to fall there between.
