Patent Application
20100120016
Avian influenza virus H5N1 was discovered in the late 1990s. Animal cases are reported in more than 46 countries and human infection is reported in 10 countries with 258 people infected and 153 deaths since 2003. Recently, the draft report of the federal government's emergency plan predicts that as many as 200 million Americans could be infected and 200,000 could die within a few months if there were an outbreak of avian influenza in the United States. An unspecified influenza pandemic is projected by the CDC to lead to about 89,000 to 207,000 deaths; 314,000 to 734,000 hospitalizations; 18 to 42 million outpatient visits; and 20 to 47 million additional illnesses in the U.S. The direct economic loss could be up to hundreds of billion dollars. A global influenza pandemic is predicted to cause between 2 million and 7.4 million human deaths worldwide.
In the United States, a recent outbreak of low pathogenic avian influenza (LPAI) in 2001 and 2002 resulted in the depopulation of over 4.5 million chickens and turkeys and is estimated to have cost the poultry industry approximately $125 million. According to the World Bank's report, by mid-2005, more than 140 million birds had died or been destroyed and losses to the poultry industry are estimated to be in excess of US $10 billion.
A key in controlling the spread of avian influenza is rapid detection of the disease followed by eradication of infected animals, quarantine within a two-mile radius to prevent movement of people and animals, and vaccination of animals outside the quarantine zone. Currently, techniques used to detect influenza, such as viral culture, RT-PCR and ELISA, are often time consuming, too expensive, or not specific to subtypes of AI viruses. Thus, a simple, rapid, robust and reliable test, suitable for use in the field or at the patient's bedside, is needed.
In one embodiment, an impedance biosensor is provided for detecting a contaminant in a starting material. The biosensor includes an input device, an output device and a microfluidic cell, all of which are supported by a housing. The starting material is engagable with the microfluidic cell and an impedance analyzer which is also supported by the housing and operable to measure impedance of the starting material to detect the presence of a contaminant.
In another embodiment, methods of detecting a contaminant in a starting material are provided. The starting material is contacted with an affinity moiety capable of binding to the contaminant to form a target. The affinity moiety is coupled to a magnetic nanoparticle. The label-free impedance bio sensor described herein is used to detect the target. Detection of the target is indicative of the presence of the contaminant in the starting material.
In yet another embodiment, methods of detecting a virus in a starting material are provided. The starting material is contacted with a red blood cell and the virus is capable of binding the red blood cell to form a complex. The complex is detected with a biosensor. Detection of the complex is indicative of the presence of the virus in the starting material.
In still another embodiment, methods of detecting a contaminant in a starting material are provided. The starting material is contacted with an affinity moiety capable of binding to the contaminant to form a target, wherein the affinity moiety is coupled to a magnetic nanoparticle. The target is detected with an impedance biosensor. Detection of the target is indicative of the presence of the contaminant in the starting material.
In another embodiment, methods of detecting a contaminant in a starting material are provided. The starting material is contacted with an affinity moiety capable of binding to the contaminant to form a target, wherein the affinity moiety is coupled to a magnetic nanoparticle. The target is separated from the starting material and delivered to a microfluidic cell. The microfluidic cell has an interdigitated array microelectrode biosensor disposed therein. The target is detected with the interdigitated array microelectrode biosensor, wherein detection of the target is indicative of the presence of the contaminant in the starting material.
Before at least one construction of the invention is explained in detail, it is to be understood that the invention is not limited to the details of construction and the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other constructions and of being practiced or being carried out in various ways.
The ability to detect the presence of small amounts of contaminants, such as bacteria and viruses, in a complex background is of vital importance to biotechnology, medical diagnosis, and prevention of pandemics. Detection and identification of contaminants in infected subjects or within the food or water supply is necessary to protect health and safety. Also, rapid detection of small amounts of contaminants will result in faster clinical diagnosis of disease, and may result in better prognosis. Detection of contaminants is difficult when only a small amount must be detected in a large sample volume or within a complex sample such as a food product or soil. There exists a need in the art for additional methods for detecting and quantifying contaminants that are sensitive, specific and rapid. In addition, there also exists a need for handheld or portable devices for detecting and quantifying contaminants which can be used in the field.
The inventors describe herein methods and systems for detection and quantification of contaminants in a starting material. A microfluidics- and interdigitated array microelectrode-based impedance biosensor is described herein. The described biosensor, which may be implemented in a portable device, allows for highly sensitive, specific, and rapid detection of contaminants and allows for minimum time between samples. Methods are also provided that allow detection of a contaminant in a starting material. The methods include contacting the starting material with an affinity moiety capable of binding to the contaminant to form a target. The affinity moiety can be coupled to a magnetic nanoparticle to form a magnetic target. The magnetic target is then detected, suitably with a biosensor such as the impedance biosensor described herein. In addition, a method is provided for detection of influenza and Newcastle viruses in a starting material by contacting the starting material with a red blood cell to form a complex and detecting the complex using a biosensor, suitably the biosensor described herein.
The biosensor technology is based on one or more concepts, including: (1) the use of magnetic nanoparticles for highly efficient and rapid separation of target virus in a poultry swab sample; (2) a microfluidic biochip with embedded interdigitated array microelectrode for precise delivery and sensitive measurement of the target virus; and (3) formation of complexes of red blood cells and nanoparticles for greater amplification of the impedance signal. Laboratory-scale experiments based on chicken swab samples that were prepared with inactivated avian influenza virus H5N1 and other viruses were completed. The results demonstrate that the biosensor is able to specifically detect avian influenza H5N1 with a limit of detection of at least 100 EID50/ml in a poultry cloacal or tracheal swab sample in less than 30 min. The biosensor will be able to operate in the field, and the cost is estimated at less than $10 per sample. The biosensor described herein introduces the concept of real time detection of avian influenza.
With reference to
With continued reference to
Referring now to
As further described below, the micro-controller 60 performs a variety of operations and functions of the biosensor 20, and communicates with various components of the biosensor 20. In one embodiment, the micro-controller 60 is a model no. AT89C55WD microchip IC offered by ATMEL Corporation. Those of ordinary skill in the art will recognize, however, that other components and combinations of components, such as microprocessors, digital signal processors, ASICs, etc. can be used in place of the micro-controller 60. For
The keypad 40 and the LCD 36 provide input and output sources for a user, respectively, although other types of input and output devices may be used. Such input and output devices are conventional in the art and will not be described further herein. For
The data storage 64 is supported within the biosensor 20 and facilitates storage of data on-board the biosensor 20. The data storage 64 can consist of a variety of conventional storage means, such as, for example a model no. AT24LC256 memory IC offered by ATMEL Corporation. It should be understood that, while the data storage 64 is shown as a separate memory, in other embodiments the data storage can be combined with other elements of the biosensor 20 (e.g., the micro-controller 60).
The communication port 80 facilitates communication between the biosensor 20 and an external electrical device such as a personal computer, printer, etc. For
The impedance detector 84 is operable to sense an impedance of samples introduced into the cartridge assembly 88 and to provide a value having a relationship with the sensed impedance to the microcontroller 60. For
The power converter 68 receives power from the power source and converts/regulates the power to the proper voltage for the circuitry shown in
The alarm 76 provides a visual and/or audible alarm. For
The power filter 72 filters the VCC voltage and includes capacitors, for
With reference to
Microfluidics technology has many advantages, among them high surface to volume ratio, small sample volume, accurate control, and low cost. A microfluidic channel (a cross section of 40 μm depth and 50 μm width) has been designed and fabricated. See Li and Su, 2006, J. Rapid Methods Automation in Microbiol 14: 96-109 (incorporated herein by reference in its entirety). An interdigitated array microelectrode (3 pairs of electrode fingers with 25 μm width) was designed and fabricated into the microfluidic channel for impedance measurement sensitive to a biological target in a sample. Impedance biosensors to detect Salmonella typhimurium and E. coli O157:H7 cells captured onto the surface of an interdigitated array microelectrode modified with specific antibody (Yang et al., 2004, Anal. Chem 76:1107-1113) (incorporated herein by reference in its entirety) or passing through an interdigitated array electrode (Varshney et al., 2007, Biosens Bioelectron, 22(11): 2408-2424) (incorporated herein by reference in its entirety) have been described.
With continued reference to
First, an interdigitated array microelectrode (“IDAM”) chip 120 is fabricated on a glass wafer 124 coated with a gold layer. In this stage, the glass wafer 124 is sputtered with 5000 Å Au with 250 Å Cr as an adhesion layer. The glass wafer 124 is then spin-coated with a 4 μm thick layer of AZ 4330 photoresist. Photolithography is used to pattern the photoresist and then the resist is developed. The Au and Cr on the glass wafer 124 is etched with the patterned photoresist as a masking layer to obtain the electrodes. In some embodiments, the IDAM chip 120 consists of 50 pairs of gold electrode fingers with a finger width of 25 μm and a gap of 25 μm between the fingers.
Second, a mold 128 is prepared for fabricating a microchannel 132. In this stage, a Silicon (Si) wafer 136 is spin-coated with SU-8 140 and soft baked. Then, the Si wafer 136 and SU-8 coating 140 is masked and UV exposed to develop the SU-8 structures 140. The mold 128 is then hard baked.
Third, the microchannel 132 is formed and bonded with the IDAM chip 120. In this stage, polydimethylsiloxane (PDMS) 144 is poured into the mold 128 and partially cured at 60° C. for 1 hour. Then, the PDMS channel 144 is pulled from the mold 128. To bond the PDMS channel 144 on the IDAM chip 120, the PDMS channel 144 is aligned with the microelectrodes 148 and thermally bonded to the chip at 80° C. for 8 hours. The resulting microchannel 132 includes an inlet 152, an outlet 156 and a detection chamber 160 therealong. In some embodiments, the microchannel 132 has a depth of 15 μm and a width of 500 μm, and the detection chamber 160 has a size of 0.5×0.5×0.02 mm and a volume of 5 nl. The microfluidic cell 108 also includes electrical connectors 164 connected to the IDAM to provide power thereto and to communicate information between the IDAM and the micro-controller 60.
Now that the biosensor 20 has been described, the purpose and exemplary operation of the biosensor 20 will be described. The biosensor 20 is operable to detect and quantify the presence of a contaminant within a sample introduced therein by utilizing impedance measurements.
The following operation of the biosensor 20 is one manner of detecting and quantifying the presence of contaminant in a sample and is not meant to be limiting. Accordingly, other manners of detecting and quantifying contaminants, for example using other biosensors, are possible and are within the spirit and scope of the present invention.
In some embodiments, the interdigitated array microelectrode operates as follows. The flow of current across the interdigitated array microelectrode, specifically across the space between the fingers, is impeded by the presence of targets in the channels. The impedance is in proportion to the amount of target that is present and is a function of the frequency of the voltage signal that is applied.
After the sample is contacted with an affinity moiety, and affinity moiety-contaminant “targets” are formed, the targets are separated from the rest of the sample and are subsequently quantified using the biosensor 20. If the affinity moieties are coupled to magnetic nanoparticles, then a magnetic separator can be used to remove the beads-plus-target complexes (see below for a further discussion of magnetic nanoparticles).
An aspect of the present system is the mechanism for contacting the targets with the interdigitated array microelectrode, specifically the use of a continuous flow system. By using a continuous flow system, multiple samples can be run through the biosensor 20 in series, with minimal downtime between samples.
Another aspect of the present system is the fact that the affinity moiety does not have to be integrated into the interdigitated array microelectrode (termed a “label-free” biosensor), but rather in some variations can be made part of the flow-through material. Because of this feature, the biosensor 20 can be reused with numerous different affinity moieties and thus can be used to detect a variety of different contaminants, without having to change microelectrodes. In other systems, by contrast, the affinity moiety (typically an antibody) is coupled to the microelectrode, with the result that the particular microelectrode can only be used to detect one contaminant. Furthermore, samples can be analyzed more rapidly with a label-free system because there is no residual material to be stripped or removed from the biosensor between readings as is the case for an “immobilized” biosensor, i.e. a biosensor having an affinity moiety attached thereto.
After the targets are introduced into the biosensor 20, oscillating voltage signals can be applied at a range of frequencies or at a single frequency, and the resulting impedances are recorded. In the absence of a positive reaction between contaminants in the sample and the affinity moiety, the affinity moieties will not form target complexes and thus will have a measurably different profile on the impedance spectrum. Conversely, when there is a positive reaction, the target complexes of affinity moieties plus contaminant that are formed will cause a measurable increase in impedance in certain portions of the frequency spectrum, producing a distinctive signature.
In a typical measurement, a sample solution is introduced into the injection port 100 and travels to the microfluidic cell 108 via the inlet tubing 104 and inlet 152 of the microchannel 132 (
Impedance measurements of the solution can be conducted by the impedance biosensor system described above or by a conventional impedance detector or analyzer, such as, for example an IM-6 impedance analyzer manufactured by BAS of West Lafayette, Ind. and with conventional software, such as, for example IM-6/THALES software. For all impedance measurements, a sine-modulated AC potential of 100 mV was applied across the IDAM and the magnitude and phase angle of impedance is measured for a frequency range from 10 Hz to 1 MHz. Using preformed magnetic nanoparticle antibody complexes (MNAC), the total detection time from sampling to measurement was 35 minutes. One pole of the IDAM chip is connected to test and sense probes, and the other pole is connected to reference and counter electrodes of the impedance analyzer.
In order to determine a frequency for the maximum difference in impedance measurements between the sample solution with attached MNAC and the control, a curve was drawn between normalized impedance change (NIC) and frequency. The value of NIC was given by the following formula (1):
where, Zcontrol is the magnitude of impedance for a control, and Zsample is the magnitude of impedance for a sample solution.
The methods described herein may be used to detect the presence of contaminants, including influenza virus, in a wide variety of starting materials with various levels of complexity in terms of antigenic diversity, density and volume. In addition to the starting materials used in the examples below, it is reasonable to expect that contaminants may be detected in a wide variety of food products, animals, and environmental and clinical samples, and may include liquid, solid or materials containing a mixture of liquids and solids. The starting materials may include vegetables, fruits, ground meats, beef, poultry, sea food, dairy, water, air, soil, blood, urine, feces; swabs from the surface of the skin, organs, trachea, or cloaca; or tissue samples. Food samples may be raw or ready-to-eat. For example, a poultry product may include a carcass, wash water from a carcass, a deboned bird, ground poultry meats, or poultry patties. The methods are also suitable for food or environmental inspection or clinical diagnosis or monitoring. For example, the methods may be used to monitor food during processing, storage, distribution or even once in the market. A solid or semi-solid starting material may be subject to homogenization prior to use in the methods.
As described in the Examples below, many types of contaminants may be detected or quantified using the methods described herein. In the Examples, Escherichia coli O157:H7 and avian influenza H5N1 were detected. In addition to these contaminants, it is reasonable to expect that one of skill in the art may use the methods with a wide variety of potential contaminants including, but not limited to, bacteria such as Listeria monocytogenes, Campylobacter jejuni, Pseudomonas mirabilis, Salmonella species and Enterococcus species; eukaryotic cells; polypeptides, including prions, toxins and blood or urine proteins; viruses such as influenza; or other chemical contaminants such as pesticides or herbicides. Significantly, both live and dead cells may be detected by the methods described herein. Starting materials, however, may be pre-treated to kill any live contaminants that may pose a health risk to a technician performing the method described herein.
Nanoparticles have previously been described. See Varshney et al., J Food Protection 68:1804-1811 (2005); Fritzsche and Taton, Nanotechnol. 14:R63-R73 (2003); Tan et al., Med. Res. Rev. 24:621-638 (2004); Zhao et al., PNAS 101:15027-15032 (2004); U.S. Pat. No. 6,623,982; and U.S. Pat. No. 6,645,731 all of which are incorporated herein by reference in their entireties. Nanoparticles range in size from 1-300 nm in diameter, suitably from 50-150 nm in diameter. Nanoparticles may have a magnetic core that may include various metals and, like microbeads, the magnetic core may be paramagnetic or superparamagnetic. Nanoparticles suitable for use in the methods described herein include those commercially available from Molecular Probes, Inc. (Eugene, Oreg.).
Nanoparticles from 50-150 nm exhibit properties of ferrofluids and remain stable colloids. They can be collected in a magnetic monolayer which allows washing away of unwanted background. Compared to other separation techniques such as centrifugation, filtration, and use of microbeads, magnetic nanoparticles provide higher capture efficiency of avian influenza virus in a poultry sample, which in turn enhances the sensitivity of the biosensor. Once the nanoparticles are contacted with the starting material and the magnetic target is formed, the magnetic target may be separated from the starting material in a variety of ways. The target may be separated by filtration or centrifugation, or by generation of a magnetic field. Magnetic separation devices suitable for use in the methods include the Magnetic Particle Concentrator (MPC) from Dynal, Inc. (Lake Success, N.Y.) and the separation system described in U.S. patent application Ser. No. 11/328,808, entitled “Separation System and Efficient Capture of Contaminants Using Magnetic Nanoparticles,” incorporated herein by reference in its entirety (
The nanoparticles may be directly or indirectly coupled to affinity moieties having affinity for the contaminant. Nanoparticles are commercially available already prepared for coupling to affinity moieties by a variety of chemical reactions, but may also be prepared by the end-user. Nanoparticles may be coupled to affinity moieties by a variety of methods including, but not limited to, pre-conjugation to streptavidin, avidin, Protein G or Protein A; using commercially available kits, nanoparticles can be directly bound to antibodies via a covalent linkage; nanoparticles with functional carboxy or amino groups exposed on their surface for use in coupling a variety of polypeptides; or nanoparticles linked to a polypeptide (a linker) capable of binding either the Fc region of an antibody, such as an Fc receptor or anti-Fc antibody, or a non-Fc region of the antibody. Polypeptides may be biotinylated by methods well known to those of skill in the art and the biotin may form a bridging complex linking a nanoparticle to an affinity moiety by binding to streptavidin or avidin. It is reasonable to expect that one of skill in the art may utilize a variety of different chemistries to couple the antibody to the nanoparticle either directly or indirectly.
The magnetic nanoparticles are able to bind to the contaminant by virtue of their coupling to an affinity moiety. Affinity moieties are suitably polypeptides that have affinity for the contaminant and will bind to the contaminant when brought into proximity with it. Affinity moieties include antibodies specific for the contaminant, ligands capable of binding a receptor on the contaminant, and receptors that bind to the contaminant. In addition to those affinity moieties exemplified below as useful in the methods described herein, it is reasonably expected that other affinity moieties with affinity for various contaminants will be suitable. Suitable antibodies may be identified using an antibody source guide, for example Linscott's Directory of Immunological and Biological Reagents or the MSRS Catalog of Primary Antibodies. Methods for generating monoclonal and polyclonal antibodies are known to those of skill in the art.
The steps of the method may be completed in several different orders. For example, the antibody may be coupled to the magnetic nanoparticle prior to contact with the starting material, or it may be added after the contaminant-antibody target is formed. Alternatively, the antibody and the magnetic nanoparticle may be added to the starting material simultaneously.
Biosensors may be utilized to detect and quantify the contaminant in the starting material. Suitable biosensors include but are not limited to, impedance biosensors, quartz crystal microbalance biosensors and viscoelastic biosensors such as that described in U.S. patent application Ser. No. 11/329,009, entitled “Method for Detecting an Unknown Contaminant Concentration in a Substance,” incorporated herein by reference in its entirety. Suitably, the biosensor described herein is utilized to detect and quantify the contaminant in the starting material.
Influenza and Newcastle viruses are capable of hemagglutinating red blood cells to form virus-red blood cell complexes. The inventors describe a method of detecting a virus in a starting material by contacting the starting material with a red blood cell. The virus in the starting material is capable of binding to the red blood cells and the complex of red blood cell-virus can then be detected using a biosensor. Suitably an impedance biosensor is used to detect the virus-red blood cell complex, more suitably the impedance biosensor described herein is utilized. The virus-red blood cell complex can be further contacted with an antibody to provide specificity to the biosensor. For use in the biosensor described herein, the antibody is coupled to a magnetic nanoparticle. As described above, the steps in the methods may be performed in a variety of orders. For example, the virus may be bound to the affinity moiety-nanoparticle complex and then incubated with red blood cells or vice versa. In an immobilized antibody-based impedance biosensor, the virus may be complexed with red blood cells prior to addition to the biosensor, or after the virus is captured in the biosensor by the affinity moiety.
In one particular embodiment illustrated in
The following examples are meant only to be illustrative and are not intended as limitations on the claims.
The following materials and methods were used throughout the Examples, unless otherwise indicated.
Frozen stock of E. coli O157:H7 (ATCC 43888) was maintained in brain heart infusion broth (BHI, Remel Inc., Lenexa, Kans.) at −70° C. The culture was harvested in BHI maintained at 37° C. for 18 to 22 h. For enumeration, pure cultures were serially diluted in 0.01 M, pH 7.4 phosphate buffered saline (PBS) and surface plated on sorbitol MacConkey (SMAC) agar (Remel Inc., Lenexa, Kans.), which was incubated at 37° C. for 20 to 22 h.
The avian influenza virus (H5N1) is produced by growth in chicken embryos and collection of allantoic fluid and killed by the National Veterinary Services Laboratory, Ames Iowa. The stock solution of virus contains approximately 1×107 egg-infectious doses (“EID”) per ml.
PBS (0.01 M, pH 7.4) was obtained from Sigma-Aldrich (St. Louis, Mo.). Bovine serum albumin (BSA; EM Science, Gibbstown, N.J.), 1.0% (wt vol−1) was prepared in PBS as a blocking buffer (PBS BSA). Protein A (from Staphylococcus aureus Cowan strain cell walls) was obtained from Sigma-Aldrich (St. Louis, Mo.). One-tenth molar (0.1 M) solution of mannitol (Sigma-Aldrich, St. Louis, Mo.) in deionized water was used for washing and resuspension of bacteria and viruses and was used for washing electrodes. All solutions were prepared with deionized water from Millipore (Milli-Q, 18.2 MΩ·cm, Bedford, Mass.).
Magnetic nanoparticles (average diameter 145 nm, 0.5 mg Fe ml−1) conjugated with streptavidin were obtained from Molecular Probes Inc. (Eugene, Oreg.). Magnetic nanoparticles have more than 85% of oxide as Fe3O4, approximately 80% wt wt−1 of magnetite, and approximately 4×1011 particles mg−1 Fe.
Affinity-purified polyclonal goat antibodies against E. coli (specific for O and K antigens) conjugated with biotin were obtained from Biodesign International (Saco, Me.). The concentration of stock solution of biotin-labeled antibodies was 4 to 5 mg ml−1. A 1:10 dilution of the antibodies was prepared in PBS (0.01 M, pH 7.4) before use.
Rabbit antibody to influenza A H5N1 Hemagglutinin (HA) (hereinafter referred to as “anti-HA antibody”) was purchased from Biodesign International (Saco, Me.). This affinity-purified antibody was raised in rabbit against a synthetic peptide (Genbank accession no. AAT76166) corresponding to 14 amino acids in the middle region of the hemagglutinin protein.
Magnetic nanoparticle antibody complexes (MNAC) were prepared in 1.7 ml sterile polypropylene centrifuge tubes. Biotin-labeled polyclonal goat anti-E. coli antibodies (7.5 μl) were continuously mixed with streptavidin-coated magnetic nanoparticles (15 μl) in 250 μl PBS BSA at 7 rpm on a variable speed rotator (ATR, Laurel, Md.) for 35 min at room temperature. Following antibody immobilization, MNAC were mixed with 150 μl of biotin solution (in PBS BSA) for 15 min to block unbound streptavidin present on the surface of magnetic nanoparticles. Excess biotin was washed out with PBS-BSA, and MNAC were resuspended in 450 μl of PBS BSA.
Serial dilutions of pure culture of E. coli O157:H7 from 8.4×102 to 8.4×108 CFU ml−1 were prepared in PBS (0.01 M, pH 7.4) buffer. A 50 μl aliquot of pure culture was mixed with 450 μl of MNAC for an immunoreaction time of 15 min. Following the immunoreaction, nanoparticle-bacteria complexes were washed three times with 0.1 M mannitol solution with an intermittent magnetic separation, and were concentrated in 100 μl of mannitol solution. Finally E. coli O157:H7 cells attached to MNAC suspended in mannitol solution was injected into the flow cell with the help of a syringe pump at a flow rate of 10 μl/min. Similar procedures were followed for viral detection.
Biotinylation of Avian Influenza (AI) H5N1 antibody was performed with EZ-Link Sulfo-NHS Biotinylation Kit (obtained from PIERCE (Rockford, Ill.)) according to the supplied instructions. Briefly, 100 μl anti-HA (1 mg/ml) was mixed with 3 μl Sulfo-NHS-Biotin solution (10 mM) into 200 μl PBS (10 mM, pH 7.4) and incubated at room temperature for 60 min. Then, excess biotin was removed by using Slide-A-Lyzer Dialysis Cassettes. The level of biotin incorporation was measured to be 4 to 5 mole biotin per mole antibody by using HABA ([(2-(4′-Hydroxyazobenzene) Benzoic Acid]) assay.
Conjugation of Magnetic Nanoparticles with Anti-HA Antibodies
Biotin-labeled H5N1 antibodies (anti-HA antibodies; 50 μl) were continuously mixed with streptavidin-coated magnetic nanoparticles (20 μl) in 50 μl PBS at 15 rpm on a variable speed rotator (ATR, Laurel, Md.) for 40 min at room temperature. After antibody immobilization, magnetic nanoparticles were mixed with 100 μl biotin solution (1 mg/ml in PBS) for 20 min to block excess streptavidin present on the surface of magnetic nanoparticles. Excess biotin was washed out by a magnetic separation.
Cloacal swab samples from birds mixed with a 10-fold serial dilution (10−2, 10−3, 10−4, and 10−5) of inactivated AI H5N1 virus (original titer was 1×107±one log EID50/ml) were suspended in 500 μl buffer (Isotonic Dextrose with Heparin: 60 grams dextrose per liter distilled water), and a cloacal swab without viruses was used as a control. The 500 μl of swab sample was then mixed with antibody-coated nanoparticles (coated with the anti-HA antibody) for an immunoreaction time of 30 min. After immunoreaction, nanoparticle-virus complexes were washed with 300 μl 0.1 M mannitol solution with an intermittent magnetic separation, and then were resuspended in 150 μl of 0.1 M mannitol solution for impedance measurement. Unless otherwise indicated, swab samples were prepared using a solution containing a virus titer of 10̂5 EID50/ml.
Cloacal and tracheal swab samples from birds were mixed with 10-fold serial dilutions (10−2, 10−3, 10−4, and 10−5) of inactivated AI H5N1 virus (original titer was 1×107±one log EID50/ml). The swabs were suspended in 2 ml buffer (Isotonic Dextrose), and cloacal or tracheal swab without viruses was used as a control.
For some experiments, it was desired to pre-coat the microelectrode with antibody to H5N1 in order to immobilize viruses on the microelectrode for subsequent analysis, using the following procedures. Firstly, Protein A (1 mg/ml in 10 mM PBS, pH 7.4) was injected into the biosensor and incubated at room temperature for ˜1.5-2 hours. Then, the microelectrode was further modified with H5N1 antibody (incubation for 2.5 hours at room temperature). After blocking with BSA (1% in 10 mM PBS, pH 7.4 and incubate at room temperature for 30 minutes), the chip was ready for a swab sample test. After each immobilization step, the microelectrode was washed with 0.1 M mannitol solution, and impedance measurements were performed.
Impedance measurement was performed using an IM-6 impedance analyzer (BAS, West Lafayette, Ind.) with IM-6/THALES software. For all impedance measurements, a sine-modulated AC potential of 100 mV was applied across the IDAM and the magnitude and phase angle of impedance were measured for a frequency range from 10 Hz to 1 MHz. Using preformed magnetic nanoparticle antibody complexes (MNAC), the total detection time from sampling to measurement was 35 min (15, 10, and 10 min for immunoreaction, washing, and measurement, respectively).
In some embodiments (including the experiments corresponding to the data shown in
One pole of the IDAM chip was connected to test and sense probes, and the other pole was connected to reference and counter electrodes of the impedance analyzer. At the end of each test, the flow cell was washed with 0.1 M sodium hydroxide for 1 h, deionized water for 30 min, 0.1 M hydrochloric acid for 1 h, and a final rinse with deionized water for 1 h. The flow rate used during washing was 10 μl/min. Mannitol solution with MNAC but no E. coli O157:H7 was used as a control for all tests.
In order to determine a frequency for the maximum difference in impedance measurements between the sample of bacterial cells with attached MNAC and the control, a curve was drawn between normalized impedance change (NIC) and frequency. The value of NIC was given by following formula:
where Zcontrol is the magnitude of impedance for a control and Zsample is the magnitude of impedance for a sample containing E. coli O157:H7. Equivalent calculations were performed for other samples including virus samples.
Ground beef was purchased from a local supermarket. A sample of 25 g of ground beef was homogenized with 225 ml of 0.1% buffered peptone water in a Whirl-pak plastic bag using a laboratory Stomacher 400 (Seward, Norfolk, UK) for 2 min. After stomaching, the sample was centrifuged two times at 250×g for 15 min in order to separate large size particles present in the ground beef stomaching water. The supernatant of the food samples was inoculated with 10-fold dilutions of E. coli O157:H7 cultures ranging from 7.9×102 to 7.9×108 CFU ml−1.
Poultry swab samples were prepared by dipping the swab, which may contain AI viruses (50-100 nm diameter), non-AI viruses, and other molecules, into Isotonic Dextrose buffer solution. Then, 1 ml of a 0.5% suspension of red blood cells was added into the swab sample solution and mixed for 1-5 min to form red cell-virus complexes. Influenza viruses, as well as Newcastle viruses, are capable of hemagglutinating red blood cells to form virus-red blood cell complexes.
The swab sample with the complexes was sent to a microfluidic channel (40 μm in depth, 100 μm in width, and 10 mm in length). An interdigitated array microelectrode (15 μm for both electrode finger width and space between electrode fingers) was embedded in the microfluidic channel, and coated with AI virus polyclonal antibodies which were bound by Protein A, producing what is referred to herein as an “immobilized” biosensor. When passed through the microfluidic channel, the AI virus-red cell complexes were specifically captured by the immobilized AI virus antibodies (anti-HA antibodies), resulting in changes in impedance. A 0.1 M mannitol wash solution was applied to wash away loosely attached non-AI molecules before the impedance was measured.
Impedances were measured using an impedance analyzer at frequencies between 1 Hz and 1 MHz. The results are depicted in
Magnetic nanoparticles (MNACs) were coupled to polyclonal antibodies specific for AI viruses (anti-HA antibodies). The nanoparticle-antibody complexes were used in the automatic magnetic sampler to separate and concentrate target viruses from a poultry swab sample. Red blood cells, which are employed as biolabels, were mixed with the captured target viruses to form the affinity moiety-coated nanoparticle-virus-red cell complex as described. The complexes were then delivered to a label-free, flow-through impedance biosensor, as described above, for impedance measurements. Changes in impedance of the affinity moiety-coated bead-virus-red cell complex were measured.
As shown in
A combination of Newcastle virus and infectious bronchitis virus was mixed in a 1:1 ratio with H5N1 avian influenza virus. The virus mixture was then optionally incubated with red blood cells and impedance was measured in a microfluidic channel with an embedded interdigitated array microelectrode on which the specific antibody had been immobilized. Impedance measurements were made using an IM-6 impedance analyzer (BAS, West Lafayette, Ind.) with IL-6/THALES software (immobilized antibody impedance biosensor).
Impedance measurements were made at various stages of coating the biosensor with reagents for antibody immobilization, as indicated. In
In
In
Tracheal swabs were obtained from chicks using standard procedures and prepared as described above. The swabs were washed into 2 ml Isotonic Dextrose buffer and centrifuged at 2000 rpm for 30 min. The supernatant from this wash was then incubated with 1 ml of a 0.5% suspension (in Isotonic Dextrose with Heparin buffer) of red blood cells for 40 min at 4° C. followed by another round of centrifugation at 1500 rpm for 10 min. The sediment was resuspended in 0.5 ml Isotonic Dextrose buffer and used for impedance analysis.
The immobilized impedance biosensor was prepared by coating the biosensor surface with Protein A, followed by anti-HA antibody and blocking with PBS-BSA. The results are shown in
Tracheal swabs were obtained from chicks using standard procedures and prepared as described above. The swabs were washed into 2 ml Isotonic Dextrose with Heparin buffer. The “Control” sample was then incubated with nanoparticle-linked anti-HA antibody complexes produced as described above. The “Tracheal Swab+H5N1” sample was supplemented with H5N1 virus. The tracheal swab was dipped into 105 EID50/ml AIV solution and subsequently washed into 2 ml Isotonic Dextrose buffer. The final concentration of virus is estimated at 104-105 EID50/ml. The resulting virus-nanoparticle-antibody complexes were then magnetically separated as described. The label-free impedance biosensor results are depicted in
Cloacal swabs were obtained from chicks using standard procedures and prepared as described above. The swabs were washed into 2 ml Isotonic Dextrose buffer, either with or without virus and centrifuged at 2000 rpm for 30 min. For those samples in which virus was added, the cloacal swab was dipped into 105 EID50/ml AIV solution, and then washed into 2 ml Isotonic Dextrose buffer. The supernatant from this wash was then incubated with 1 ml of 0.5% red blood cells (in Isotonic Dextrose with Heparin buffer) for 40 min at 4° C. followed by another round of centrifugation at 1500 rpm for 10 min. The sediment was resuspended in 0.5 ml Isotonic Dextrose buffer and used for impedance analysis.
The immobilized impedance biosensor was prepared by coating the biosensor surface with Protein A, followed by anti-HA antibody, and finally blocking with PBS-BSA as described above. The results are shown in
Cloacal swabs were obtained from chicks using standard procedures and prepared by exposing the swabs to various concentrations of H5N1 influenza virus. The cloacal swabs were dipped into a series of ten-fold dilutions of AIV solution and subsequently washed into 2 ml Isotonic Dextrose buffer. The swabs were washed into 2 ml Isotonic Dextrose with Heparin buffer and centrifuged at 2000 rpm for 30 min. The supernatant from this wash was then incubated with 1 ml of 0.5% red blood cells (in Isotonic Dextrose with Heparin buffer) for 40 min at 4° C. followed by another round of centrifugation at 1500 rpm for 10 min. The sediment was resuspended in 0.2 ml Isotonic Dextrose with Heparin buffer and used for impedance analysis.
The immobilized impedance biosensor was prepared by coating the biosensor surface with Protein A, followed by anti-HA antibody and blocking with PBS-BSA as described above. The results are shown in
Cloacal swabs were obtained from chicks using standard procedures and prepared by exposing the swabs to various concentrations of H5N1 influenza virus. The cloacal swabs were dipped into a series of ten-fold dilutions of AIV solution, at 1/100, 1/1000, 1/10000, and 1/100000 final concentrations, and subsequently washed into 2 ml Isotonic Dextrose buffer. The swabs were then incubated with nanoparticle-linked anti-HA antibody complexes produced as described above. The resulting virus-nanoparticle-antibody complexes were then incubated with 1 ml of a 0.5% suspension of red blood cells (in Isotonic Dextrose with Heparin buffer), and subsequently magnetically separated as described above.
The label-free impedance biosensor results are depicted in
Avian influenza H5N1 was also detected using the magnetic nanoparticle-antibody complexes and the label-free impedance biosensor described herein. Briefly, streptavidin-coated nanoparticles were coupled to biotinylated anti-HA antibodies and either H5N1 influenza or a mixture of Newcastle virus and infectious bronchitis virus were added to the nanoparticle-antibody complex. The complexes were passed through the impedance biosensor. The results, depicted in
As shown in
Another set of virus-detection experiments was conducted using a non-commercial polyclonal anti-H5 antibody generated in rabbit. The antibody was raised against recombinant H5 polypeptide (obtained from Protein Science Inc., Meriden, Conn.; A/Vietnam/1203/2004) using standard protocols. Anti-H5 antibodies from the rabbit were purified by ammonium sulphate precipitation, dialyzed against acetate buffer and phosphate buffer and made available for biosensor applications.
In some experiments (results shown in
The anti-H5 antibodies were biotinylated to facilitate attachment to streptavidin-coated nanoparticles. Biotinylation of anti-H5 antibody was performed with EZ-Link Sulfo-NHS Biotinylation Kit according to the supplied instruction. Briefly, 100 μl anti-HA (4.9 mg/ml) was mixed with 3 μl Sulfo-NHS-Biotin solution (10 mM) into 200 μl PBS (10 mM, pH 7.4) and incubated at room temperature for 60 minutes. Then, excess biotin was removed by using Slide-A-Lyzer Dialysis Cassettes. The level of biotin incorporation was measured to be 4 to 5 mole biotin per mole antibody by using HABA assay.
Biotin-labeled anti-H5 antibodies (50 μl) were continuously mixed with streptavidin-coated magnetic nanoparticles (20 μl) in 50 μl PBS at 15 rpm on a variable speed rotator (ATR, Laurel, Md.) for 40 min at room temperature. After antibody immobilization, magnetic nanoparticles were mixed with 100 μl biotin solution (1 mg/ml in PBS) for 20 min to block excess streptavidin present on the surface of magnetic nanoparticles. Excess biotin was washed out by a magnetic separation.
Ten-fold serial dilutions (10−2, 10−3, 10−4, 10−5, and 10−6) of inactivated AI H5N1 virus (dilutions of a solution in which the original titer was 1×107±one log EID50/ml) were suspended in 500 μl buffer (Isotonic Dextrose), and buffer without virus was used as a control. The 500 μl of sample was then mixed with antibody coated nanoparticles for an immunoreaction time of 30 min. After immunoreaction, nanoparticle-virus complexes were washed with 300 μl 0.1 M mannitol solution with an intermittent magnetic separation, and then were resuspended in 150 μl of 0.1 M mannitol solution for impedance measurement.
The antibody was attached to the biosensor using protein A. Firstly, Protein A (1 mg/ml in 10 mM PBS, pH 7.4) was injected into the microfluidic chamber and incubated at room temperature for 1.5-2 hours. Then, the microelectrode was further modified with anti-H5 antibody (245 μg/ml) (incubation for 2.5 hours at room temperature). After blocking with BSA (1% in 10 mM PBS, pH 7.4 and incubate at room temperature for 30 minutes), the chip was read for a sample test. After each immobilization step, the microelectrode was washed with 0.1 M mannitol solution, and impedance measurements were performed.
Impedance measurements were performed using an IM-6 impedance analyzer (BAS, West Lafayette, Ind.) with IM-6/THALES software. For all impedance measurements, a sine-modulated AC potential of 100 mV was applied across the IDAM (interdigitated array microelectrode) and the magnitude of impedance and phase angle were measured for the frequency range from 10 Hz to 1 MHz.
Results of impedance measurements are shown in
This application claims priority to provisional application No. 60/841,774, filed Sep. 1, 2006, and to provisional application 60/876,919, filed Dec. 22, 2006, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US07/77376 | 8/31/2007 | WO | 00 | 12/2/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60841774 | Sep 2006 | US | |
| 60876919 | Dec 2006 | US |
