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Project: An Immunochromatographic Strip for Tasting Rotavirus

Learning Individual: Milton Young

Course: Nanotechnology and Nanosensors

Instructor: Professor Hossam Haick

Coursera: June 2015

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Biography

Milton Young is head of operations for a micro cooling, heating and power generation (m-

CCHP) startup in Houston, Texas, United States. His areas of expertise include manufacturing

operations, global sourcing, project planning, ERP systems implementation, product

development and finance. He previously worked at McCoy Global, FMC Technologies,

Whirlpool, ExxonMobil and General Motors. He holds an MBA from The J.L. Kellogg School of

Management at Northwestern University (USA), an MS in Chemical Engineering from Yale

University (USA) and a BS in Chemical Engineering from Mississippi State University (USA).

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Table of Contents

Abstract …………………………………………….………………………………………….…. 4

Introduction …………………………………………………………………………………….... 5

Literature Review …………………………………………………………………………….….. 6

Project Description ……………………………………………………………………………… 8

Design Method for Fabrication ………………………………………………………………… 8

Design Method for Application ………………………………………………………………… 9

Conclusions and Recommendations …………………………………………………………. 11

References .……………………………………………………………………………………… 12

Figures

Figure 1. Schematic of assembled ICG strip and principle of detection. (Adapted from Feng

2015, with rotavirus antibodies) ……………………………………………………………….. 10

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Abstract

Rotavirus is a contagious, water-born pathogen that is the leading cause of disease among

children. Concentrations small enough to infect are too small for most traditional detection

methods. These traditional methods are time consuming, require expensive analytical

equipment and utilize stool samples from infected (or likely infected) people. The unique

properties of nanomaterials make them attractive for designing biosensors with high specificity

to pathogens, like rotavirus, quicker reaction times and simple visualization of results. A

method is proposed to develop an immunochromatographic (ICG) strip to capture and detect

rotavirus antigens utilizing rotavirus antibodies conjugated on to gold nanoparticle immobilized

on a cellulose strip. This method is analogous to tasting rotavirus and translating the resulting

taste into a visual representation, a red color on the test strip. Specimens can be collected be

prior to ingesting suspected water or hand-to-moth contact. The proposed method may also

lead to using nanomaterials to capture rotavirus and other pathogens at point of use.

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Introduction

Rotavirus is the leading cause of disease among children. It is a contagious, water-borne virus

primarily transmitted orally. Once ingested, rotavirus is resistant to the strong pH of the

stomach and the digestive enzymes in the gastrointestinal tract (Epitope Diagnostics 2015).

Rotavirus infections are one of the most common reasons for hospitalizations due to

gastrointestinal (stomach flu) diseases (Mezger 2014). As a pathogen, rotavirus is hard to

detect with human senses. Being able to “taste” rotavirus might enable people to avoid it.

This project proposes to create an immunochromatographic tongue, based on existing

nanotechnologies, to taste and see rotavirus.

Rotavirus

Rotavirus is a genus of a virus in the family Reoviridae. There are five species of Rotavirus,

referred to as A, B, C, D, and E. The most common species, Rotavirus A, causes more than

90% of infections in humans (Fitzgerald 2015). Six viral proteins (VP1, VP2, VP3, VP4, VP6 and

VP7) form the virus particle (virion). Once cells are infected, rotaviruses produce six

nonstructural proteins (NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6) (Fitzgerald 2015).

Rotaviruses have a genome consisting of 11 double-stranded RNA segments surrounded by a

distinctive three-layered icosahedral protein capsid (Epitope Diagnostics 2015) formed by the

different viral proteins. Viral particles are up to 100nm in diameter (Epitope Diagnostics 2015).

Concentration levels, incubation and transmission

Rotavirus occurs when fecal coliform seeps into groundwater where people use rivers for

bathing or perform body functions near drinking water sources. Infections can be caused by

concentrations as small as 10 focus forming units (10 FFU) per milliliter of water (USA EPA

2010). In comparison, groundwater and relatively clean surface water may have

concentrations of 1 rotavirus/100L (Dore 2014). Due to the infectious nature of rotavirus,

detection in range of 10 to 100 organisms per several liters of sample is required for large scale

water monitoring (Pires 2014). Rotavirus takes 2 days to incubate and 3 days for symptoms

(vomiting and water diarrhea) to show (National Vaccine Information Center n.d.). In

developing countries direct transmission often comes from hand-to-mouth transmission

(Mattioli 2015).

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Taste

Taste is one of the five main human senses. It is the sensation produced when a substance in

the mouth reacts chemically with any of the million taste receptor cells located on taste buds

on the tongue. Taste senses both beneficial and harmful things; sweetness helps identify

energy-rich foods, while bitterness warns of toxins (Miller 2011). Many plants with poisonous

compounds have a bitter taste. This bitter taste warns humans and animals that the item

potentially can cause harm and should be avoided, much as bright color warns animals and

humans to avoid eating certain plants and animals. For example, certain frogs secrete a

protein substance which when tasted tells the taster not to eat them. Once an animal has

tasted the unpleasant poison of a Dendronbatidae they tend to avoid it in the future; this

technique is called aposematic coloration (Wilderness Classroom 2008). The ability to taste a

particular substance depends on the type of food and the concentration. People can taste

sour, like lemons, in concentrations of mg/liter. Human taste testers have been used

throughout history to detect poisons such as arsenic trioxide, cyanide and strychnine (Luthern

2009). Tasting works if the samples are large enough to trigger taste buds or an immediate

chemical or allergic reaction in the taster.

Why taste instead of smell or sight?

Concentrations of rotavirus that cause illness are too small to see, smell or taste. Furthermore,

rotavirus is only dangerous after it is ingested. Once in the mouth, taste might determine if the

sample is good or bad. If we can develop a “taste” signal and convert it into a visual signal to

supplement the tongue, preferably before rotavirus is ingested, then we have a means of

avoiding rotavirus.

Literature review

Current Methods for detecting Rotavirus

Most methods for detecting microorganisms like rotavirus are employed at the source rather

than point of use. Well-established detection methods include: cell cultures, immunological

methods, polymerase chain reactions (PCR), and electron microscopy identification (Mezger

2014, Samendra 2014). These methods often take days to obtain results (Samendra 2014) or

require expensive and complex equipment or elaborate sample preparation. Despite the

relatively high cost of equipment, microarray-based detection methods are highly attractive

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due to their high sensitivity and rapidity and because these DNA/RNA microarrays are an

important tool in gene expression studies, genotyping, pharmacogenomics, pathogen

classification, drug discovery, sequencing and molecular diagnostics (Rodrigo 2014). The

most widely used rotavirus diagnosis methods detect protein antigens on rotavirus particles in

stool specimens after the particles have been ingested and has the opportunity to incubate.

What’s needed is a biosensor to detect rotavirus in samples before it has opportunity to enter

the human system and incubate.

A biosensor is an analytical device that converts molecular recognition of a target analyte into a

measurable signal via a transducer (Sin 2014). Nanotechnology has merged with biosensing to

improve sensitivity and detection limits of biological and chemical events. Improved sensitivity

and detection is due to the greater surface area of nanostructure sensing surfaces, such as

carbon nanotubes, nanowires, graphene, gold film and conductive polymers incorporated into

the conducting transducers (Syed 2014) and the ability to form complex structures which can

hold antigens, antibodies and aptamers and have interesting color properties (Haick 2015).

Pathogenic sensing relies on either immunosensing or nucleic acid detection. Immunosensors

are based on the interaction between antigens on the target cells and antibodies immobilized

on capture and detection surfaces. The resulting conjugates have been detected via various

sensing methods, including fluorescence, electrical or electrochemical impedance, cantilever,

quartz crystalline microbalance (QCM), or surface plasmon resonance (SPR) (Samendra 2014).

Several methods to detect rotavirus with nanomaterials have been developed within the last

five years. Three promising methods use the color or fluorescence quenching properties of

nanoparticles: 1) a colorimetric immunoassay using magnetic and platinum nanoparticles

entrapped in large pore-sized meso-cellular carbon which produces a blue color (Kim 2014), 2)

an immuno-biosensor in which rotavirus is captured by rotavirus-specific antibodies

immobilized on a graphene oxide (GO) array with detection observed by the fluorescence

quenching that results from fluorescence energy transfer between GO and gold particles linked

to the antibodies (Jung 2010) and 3) a proposed lateral flow immunochromatographic (ICG)

strip in which monoclonal antibodies conjugated onto gold nanoparticles are used to detect

pathogens and a second monoclonal antibody is used to capture the detected pathogens

(based on Feng 2015). The three methods listed here are all indirect; they use a primary

antibody for locating the antigen and a second antibody to detect the primary antibody. The

secondary antibody is usually polyclonal and amplifies the signal over the single antibody

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(direct) method (Fitzgerald 2015). Of the three, the ICG strip has higher potential for a point-

of-use application.

Project description - overall design method for fabrication and application

The project described here utilizes the ICG strip method adapted for rotavirus antibodies. The

ICG strip method has been successfully demonstrated to detect melamine (Sun 2012) and

Pantoea stewartii Stewartii (Feng 2015). The ICG strip has also been used to detect lead in

water samples (Kuang 2013).

Design method for fabrication

Monoclonal and Polyclonal Antibody (mAb and pAb)

Taniguchi, Urasawa and Urasawa (1985) first established methods to prepare and characterize

human rotavirus monoclonal antibodies (mAb). Several companies now sell rotavirus

antibodies. Abcam (2015) offers 6 antibodies with 1 (ab20036) suitable for conjugation.

Thermo Scientific (2015) offers 16 antibodies of which 10 are monoclonal and 3 are suitable for

conjugation. Fitzgerald Industries International's (FII) rotavirus antibody is a mouse monoclonal

antibody (clone M74252). FII’s rotavirus antibody was generated using rotavirus as the antigen.

This antibody has been shown to work in applications such as: ELISA. FII has three mouse

monoclonal VP6 antibodies, which are reactive to the VP6, VP7 and VP8 rotavirus proteins

(Fitzgerald 2015). The VP6 antibody (10-1021) is a capture antibody, and the VP7 antibody

(10-1022) is a detection antibody. The mAb combinations will be assessed by sandwich ELISA

and used as capture antibody and gold-labeled antibody, respectively, in the ICG strip.

Colloidal Gold Nanoparticles (adapted from Sun 2012)

Colloidal gold particles will be prepared by first heating to boiling 200 mL of 0.1 g/L chlorauric

acid under constant stirring (100 g), then mixed with 8.0 mL of 1% trisodium citrate (w/v) at 300

°C, and stirred for 10 min until the color of the solution turned from yellow to wine-red. The

solution will be allowed to cool at room temperature under constant stirring and stored at 4 °C.

All solvents will be prepared with deionized water. Transmission electron microscopy (TEM)

examinations will be used to verify that the gold nanoparticles have uniform particle size

(approximately 30 nm in diameter).

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Gold Nanoparticle-Labeled mAb (adapted from Feng 2015, Note: All concentrations are for

example purposes only and must be verified.)

Through the negative charge on surface, colloidal gold particles can quickly and steadily

adsorb the positively charged polymer material such as proteins without destroying its

biological activity. The ion concentration and the ratio of both materials will impact the

adsorption of colloidal gold to proteins. The colloidal gold solution will be adjusted to pH 7.0

with 0.1 M K2CO3. Subsequently, 0.16 mg mAb in phosphate buffer solution (PBS) at pH 7.4

will be added drop by drop into 10 mL colloidal gold nanoparticle solution and kept at room

temperature for 50 min. One milliliter of 0.5% BSA (w/v) will be added slowly into the solution

to block the gold nanoparticles and stabilize the labeled mAb. Following a 2-hour incubation,

the solution will be centrifuged at 7000 g for 30 min. The resulting precipitate will be washed

three times with 0.02 M PBS (containing 5% sucrose, 1% BSA, and 0.5% PEG 6000, pH 7.4),

and dissolved in 5 mL of 0.02 M PBS (containing 0.02% NaN3), and stored at 4 °C.

ICG Strip Preparation (adapted from Feng 2015, Note: All concentrations are for example

purposes only and must be verified.)

The ICG strip consists of four parts: the sample pad, the nitrocellulose (NC) membrane, the

polystyrene backing card, and the absorption pad, which will be assembled in layers. The

capture mAb will be used in the test line (T line) to detect the presence of pathogens in the

samples; goat anti-mouse IgG will be used in the control line (C line). These immunoglobulins

will be sprayed onto the nitrocellulose (NC) membrane at 1 μL/cm using a membrane dispenser

(XinqidianGene-Technology Co. Ltd., Beijing, China) at the concentration of 4 mg/mL to

capture mAb and 0.5 mg/mL to the goat anti-mouse IgG, respectively. The NC membrane will

be dried at 37 °C for 30 min. The sample pad will be immersed in PBS (containing 1% BSA and

0.2% Tween 20) and dried at 37 °C for 4 h to minimize nonspecific binding and matrix

interference. The assembled strips will be placed into a plastic drum prior to use.

Design method for application (Adapted from Feng 2015)

This ICG assay will be based on an antibody pair: one type of mAb will be conjugated to gold

nanoparticles (GNPs) as the detection antibody and the other type of mAb or pAb will be

sprayed onto the NC membrane as the capture antibody. Prior to the test, GNP-mAb will be

added to the sample and allowed to react at room temperature for 5 min. The reaction solution

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is then added to the sample pad of the strip. After 5 min, the results will be observed with the

naked eye. If the sample contains pathogens, GNP-mAb binds to the pathogens. Through

capillary action, the reaction solution will flow to the absorption pad, where the capture

antibody immobilized at the T line will capture the GNP-mAb-pathogens. With the deposition of

GNP-mAb-pathogens, a red line will appear on the NC membrane (Figure 1A).

A sample will be positive for rotavirus if two lines appear: T and C. A sample will be negative

for rotavirus if only the C line appears (Figure 1B). The intensity of the T line reflects the amount

of captured GNP-mAb-pathogens. The more pathogens are captured, the greater the GNP-

mAb interaction and the higher the color intensity on the T line. The C line should always

appear; otherwise, the procedure was incorrectly carried out or the strip was invalid and a

repeat test with a new strip should be performed.

End User instructions

Determine what is to be sampled. If the sample is to be taken from the hands, first rinse hands

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in basin of clean water, then pour the water into a container, and add the GNP-mAb solution to

the container. Finally dip the ICG strip into the water in the container up to the sample pad

line, pull out the strip, let the strip dry, then wait several minutes for antibody binding and

detection to occur. If the sample is from a water source, pour a sample into a container and

add the GNP-mAb solution to the container. Finally dip the ICG strip into the water sample in

the container, up to the sample pad line, pull out the strip, let the strip dry, wait several minutes

for antibody binding and detection to occur. In either case the presence of rotavirus results in

color change.

Conclusions and recommendations

Based on the latest research on application of nanomaterials to detecting pathogens, I have

proposed a simple and effective biosensor tongue, which can be used to taste and see

rotavirus, the leading cause of disease among children. The capture and detection

mechanisms are well understood. Fabrication is straightforward with materials either

commercially available or easy to fabricate. Further development and investigation is needed

to determine costs of mass production, distribution, and storage for the GNP-mAb solution

and the biosensor tongue (ICG strip). Individual and public responses (actions) also need to be

developed on what to do with potentially infected people if rotavirus is discovered.

Recommendations for future work include: identifying the common characteristic of the most

pressing types of rotavirus, improving detectability at lower concentrations, developing an

aptamer to supplement antibody and combining detection of multiple pathogens onto the

same tasting strip. With additional research into the capture mechanism, it may be possible to

develop a nanomaterial filter to capture rotavirus on a personal, usable scale, perhaps quart or

liter quantities in minutes.

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References

Abcam (2015).

http://www.abcam.com/products?keywords=Rotavirus&selected.productType=Primary+antibo

dies&selected.targetName=Rotavirus. Accessed June 4 2015.

Biocompare (2015). http://www.biocompare.com/9776-Antibodies/604730-Rotavirus-

antibody/#. Accessed June 4 2015.

Centers for Disease Control & Prevention (n.d.). Rotavirus. http://www.cdc.gov/rotavirus/. Accessed June 6 2015. Dore, M. H. (2014). Global Drinking Water Management and Conservation: Optimal Decision-Making, Springer International Publishing. Epitope Diagnostics. http://www.epitopediagnostics.com/ktr841/. Accessed June 2 2015 Feng, M., Kong, D., Wang, W., Liu, L., Song, S., & Xu, C. (2015). Development of an Immunochromatographic Strip for Rapid Detection of Pantoea stewartii subsp. stewartii. Sensors, 15(2), 4291-4301.

Fitzgerald International Industries (2015). Guidelines Colloidal Gold Assays. https://www.fitzgerald-fii.com/media/Protocols/FII_Colloidal_Gold_Guidelines.pdf. Accessed June 5 2015.

Fitzgerald International Industries (2015). https://www.fitzgerald-fii.com/catalogsearch/result/?q=Rotavirus+VP6+antibody. Accessed June 5 2015.

Gentsch, J. R., Glass, R. I., Woods, P., Gouvea, V., Gorziglia, M., Flores, J., ... & Bhan, M. K. (1992). Identification of group A rotavirus gene 4 types by polymerase chain reaction. Journal of Clinical Microbiology, 30(6), 1365-1373.

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Mattioli, M. C. M., Davis, J., & Boehm, A. B. (2015). Hand-to-mouth contacts result in greater ingestion of feces than dietary water consumption in Tanzania: A quantitative fecal exposure assessment model. Environmental science & technology. Mezger, A., Öhrmalm, C., Herthnek, D., Blomberg, J., & Nilsson, M. (2014). Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification. PloS one, 9(11), e111874. Miller, G. (2011). Sweet here, salty there: evidence for a taste map in the Mammalian Brain. Science, 333(6047), 1213. Minnesota Rural Water Association, Minnesota (2009). Department of Health, Minnesota Training Coalition, United States. Office of Drinking Water, Minnesota Water Works Operations Manual. Minnesota Rural Water Association, Fourth Publication Date: Summer 2009. http://www.mrwa.com/mnwaterworksmnl.html. Accessed May 23 2015 National Vaccine Information Center (n.d.). Rotavirus and Rotavirus Vaccine. http://www.nvic.org/vaccines-and-diseases/Rotavirus.aspx. Accessed May 23 2015. Pires, N. M. M. (2014). Integratable opto-microfluidic devices for sensitive detection of bio-analytes. Rodrigo, M. A. M., Zitka, O., Krejcova, L., Hynek, D., Masarik, M., Kynicky, J., ... & Kizek, R. (2014). Electrochemical Microarray for Identification Pathogens: A. Int. J. Electrochem. Sci, 9, 3431-3439. Samendra, P. S., Masaaki, K., Charles, P. G., & Ian, L. P. (2014). Rapid Detection Technologies for Monitoring Microorganisms in Water. Biosens J, 3(109), 2. Sin, M. L., Mach, K. E., Wong, P. K., & Liao, J. C. (2014). Advances and challenges in biosensor-based diagnosis of infectious diseases. Expert review of molecular diagnostics, 14(2), 225. Sun, F., Liu, L., Ma, W., Xu, C., Wang, L., & Kuang, H. (2012). Rapid on‐site determination of melamine in raw milk by an immunochromatographic strip. International Journal of Food Science & Technology, 47(7), 1505-1510. Syed, M. A. (2014). Advances in nanodiagnostic techniques for microbial agents. Biosensors and Bioelectronics, 51, 391-400. Taniguchi, K., Urasawa, S., & Urasawa, T. (1985). Preparation and characterization of neutralizing monoclonal antibodies with different reactivity patterns to human rotaviruses. Journal of general virology, 66(5), 1045-1053. US EPA (2010). Quantitative Microbial Risk Assessment to Estimate Illness in Freshwater Impacted by Agricultural Animal Sources of Fecal Contamination. Office of Water. Dec 2010. Wikipedia (n.d.). Taste. http://en.wikipedia.org/wiki/Taste. Accessed June 6 2015.

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Wilderness Classroom (2008). Poison dart frogs. http://www.wildernessclassroom.com/wilderness-library/poison-dart-frogs/. Accessed June 6 2015. World Health Organization (2009). Manual of rotavirus detection and characterization methods.