Pathogen Biosensors

Randy Yao, Samantha Williams

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Pathogens (GERMS)

• Pathogens are: anything that causes disease in its host.• Examples: Bacteria, Virus, Fungus, Prion• More well known as "germs".

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Presentation Overview

1. Intro/Lecture Review… ……………………………………….. 4

2. Why do we need biosensors?..................................... 9-specific case studies

3. Ideal Pathogen Biosensor …….……………………………… 15

4. Brief History …………………………………………………….. 17

5. Current pathogen biosensors ……………………………… 19-E. Coli - Staphylococcus

6. Problems with Current Biosensors ……………………… 26

7. Looking to the Future ……………………………………..… 28

8. Conclusion ……………………………………………………….. 34

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Lecture Review

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Biosensors Lecture (14) ReviewBiosensors usually have three parts:• A receptor (recognizes interested molecule)• A transducer (converts the recognition into a signal)• (optional; $) A data processor (ADC - processes the

signal into an interface favorable for presentation to the customer)

Example: Blood glucose biosensor

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Quiz Time - Mix, Match and Order

A. Transducer

B. Data Processor

C. Bioreceptor

Something that only interacts with the interested molecule, and does not decay during the interaction.

Converts the signal generated to an user friendly interface.

Recognizes the interaction between the receptor and the analyte, and transforms it into a signal measurable. (e.g. current , heat)

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C

B

A

The "3EJ4 Block Diagram" For Biosensors

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A look at specific case studies

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Pathogen Biosensors: Why We Need Them

• Safety detection meat productso Mad Cow Disease

• Have Diary Products gone bad?

• A possible protection against biological warfareo Rampant throughout human history. (e.g. Sino-Japanese

War (WWII), Korean War, Vietnam War, World War I mustard gas, etc .)

o Pathogens are spread throughout enemy territory to decrease their ability to support front-line troops in prolonged conflicts.

o Biosensors can be great at discouraging this as well.

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Any Problems?

Reminder: the bio-receptor usually has to react specifically to the interested molecule (analyte), and should not decay.

Specificity!

Solution to problem - A big array of biosensors (60- 80) are used to probe the DNA sequence.

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Specific Cases Call for Biosensors

2008 - Milk power with overtly concentrated Melamine

• WHO - "Melamine can be found in milk at low ppb to very low ppm ranges"

• Milk powder was tested up to contain 691 ppm.

• 6-11 toddler deaths due to kidney damage• 1000+ infants hospitalized• 300,000-600,000 victims

Currently biosensors are helping people detect concentrated melamine and prevent these deaths.

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"Melamine.. was added to the milk to cause it to appear to have a higher protein content."

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• What Happened?[1]▫ Contamination of deli meats▫ Pulled about 220 products ▫ Cost Maple Leaf about $20

million▫ Twenty people died and many

others became ill

Maple Leaf Listeria Contamination (2008)

• Currently, there are no rapid, inexpensive techniques to warn that something could be wrong [2]

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Ideal Requirements:

Accuracy False-positive and false-negative results must be low or preferably zero

Assay Time The biosensor should produce a ‘real-time’ response

Sensitivity Failure to detect false-negative results, lowers the sensitivity of the assay

Specificity The biosensor should easily discriminate between the target organism or toxin and other organisms

Reproducible Each assay should be highly reproducible and easy to calibrate

Robust The biosensor must be able to resist changes in pH, ionic strength and be sterilisible

User Friendly The assay should be fully automated and require minimal operator skills for routine detection

Compatible Interface

The biointerface should be compatible with the transduction principle, resist non-specific binding and should be freely accessible in three- dimensional space

Validation The biosensor assay should be evaluated against current standard techniques and a LOD obtained

Table 1: Ideal requirements for biosensor-based microbial detection assay [3]

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Brief History

1956 - Professor Leland C Clark defines biosensor

1962 - "Landmark address" at the New York Academy of Science to make "electrochemical sensors"

1975 - (First Generation) blood glucose biosensor becomes a commercialized reality.

1990 - Commercialized pathogen biosensor first launched by Pharmacia BiosensorAB for analysis for bacteria and larger "whole cells".

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E. Coli and Staphylococcus

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• Can be ingested from contaminated water, unpasteurized milk, and beverages.

• Common symptoms include:▫ severe stomach aches ▫ diarrhea

Current Method:

• Bioreceptor - antibodies that attach themselves to E Coli

• Transducer - a rod that vibrates at different frequencies when the antibodies are attached to E Coli

Video: http://www.sciencedaily.com/videos/2006/1109-e_coli_handheld_sensor.htm

E Coli Biosensors

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Staphylococcus aureus

• Spherical bacterium • One of the most common

food-borne diseases world- wide [4]

• Found on skin and in noses of ~25% of healthy people [5]

• Capable of producing a heat- stable toxin (enterotoxin) that can cause gastrointestinal illness [6]

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[7]

• Common Symptoms of Staph Food Poisoning [ 5] :

▫ Nausea▫ Vomiting▫ Abdominal cramping▫ Dehydration

• In severe cases, can cause toxic shock syndrome or changes in blood pressure and heart rate

Staphylococcus aureus

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Surface Plasmon resonance-based biosensors (SPR)

• Biomolecular recognition element (usually an antibody) is immobilized on the surface of the sensor [8]

• Interaction of staphylococcal enterotoxin B (SEB) and antibody results in a thin biomolecular layer which changes the refractive index

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[3]

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[9]

Benefits of SPR

• Allows for continuous real-time analysis [9]

• Detection does not consume reagents unlike the majority of other procedures such as ELISA [9]

• Can work in an amplification mode (in addition to a direct detection mode) [9]

• Commercial SPR-based Biosensors: [3]

▫ GE - BIAcore system▫ Artificial Sensing Instruments - BIOS-1▫ Texas Instruments - SPREETA

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Problems with Current Biosensors

• Still lack the ‘real time response’ needed for emergency situations and perishable food items etc.

• Require sophisticated instrumentation and skilled operators – difficult to use in remote settings or developing countries

• Significant sample pre-treatment

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Bioactive Paper

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Bioactive Paper• Research being done at McMaster

• Bioactive paper: “paper-like products, cardboard, fabrics and their combinations etc., with active recognition and/or functional material capabilities”[10]

• Pros of using Bioactive Paper over traditional biosensors [11]:▫ Inexpensive ▫ No need for laboratories or sophisticated instrumentation▫ Rapid response times

• How it works:▫ Special proteins (Antibodies) attached to the structure of

the paper to bind pathogens and other contaminants [12]

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How can Bioactive paper be used?

• Food-Borne Illness▫ Paper-towel that can detect the presence of

bacteria on food preparation surfaces [12]

• Water Contamination▫ Bioactive paper strip that can remove pathogens

and indicate that the water is safe [12]

• Health Care▫ Face masks that actively bind viruses to the filter

surface [12]

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Bioactive Paper Sensor for Detection of Neurotoxins

• Detection of AChE inhibitors• Paper-based solid-phase biosensor▫ Uses “piezoelectric inkjet printing of biocompatible,

enzyme-based sol-gel-based inks to create colorimetric sensor strips” [13]

• Measures residual activity of AChE

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[ΰ]

Ellman Colorimetric Assay

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[13]

Figure 1: Dose-dependent inhibition of acetylcholinesterase by various concentrations of paraoxon. Inset is the dose-dependent inhibition responses with lower levels of paraoxon. [13]

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References[1] R.Rampton. (2008). Canada says 12 dead in food poisoning outbreak,” Reuters.

[Online] Available: http://www.reuters.com/article/2008/08/26/us-meat- idUSN2526525120080826

[2] Sentinel (2011). “SENTINEL Bioactive Paper Network,” SENTINEL. [Online]. Available:

http://www.nsercpartnerships.ca/How-Comment/Networks-Reseaux/SENTINEL- SENTINEL-eng.asp

[3] P.Leonard et al “Advances in biosensors for detection of pathogens in food and water,” Enzyme and Microbiology Technology, vol. 32, pp. 3-13. 2003.

[4] A. Ostyn at al. (2010). “First evidence of a food poisoning outbreak due to staphylococcal enterotoxin type E, France, 2009,” Eurosurveillance. [Online]. Available:

http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19528

[5] Center for Immunization and Respiratory Diseases. (2006). “Staphylococcal Food Poisoning,” Centers for Disease Control and Prevention. [Online]. Available: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/staphylococcus_food_g.htm

[6] Defending Food Safety. (2011) “Staphylococcus Fact Sheet,” Defending Food Safety. [Online]. Available: http://www.defendingfoodsafety.com/2008/12/food- safety-law/common-food-borne-pathogens/staphylococcus-fact-sheet/

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References[7] M. Conrad Stroppler. (2011). “Staph Infection (Staphylococcus aureus),” Medicine Net.

[Online]. Available: http://www.medicinenet.com/staph_infection/article.htm#

[8] J. Homola at al. “Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk,” International Journal of Food Microbiology, vol. 75, pp. 61-69. 2002.

[9] A.N. Naimushin at al. “Detection of Staphylococcus aureus enterotoxin B at femtomolar levels with a miniature integrated two-channel surface plasmon resonance (SPR) sensor,” Biosensors and Bioelectronics, vol. 17, pp. 573-584. 2002.

[10] S. Aikio, S. Gronqvist et al. Bioactive paper and fibre products patent and literary survey, VTT Working Paper 51, VTT Technical Research Center of Finland, Oulu, Finland, 2006.

[11] R.Pelton, “Bioactive Paper Provides a Low-Cost Platform for Diagnostics,” Trends in Analytical Chemistry, vol. 28, no. 8. Nov 8, 2009

[12] Sentinel (2011). “What is Bioactive Paper?,” SENTINEL. [Online]. Available: http://www.bioactivepaper.ca/index.php?module=page&id=6000

[13] S.M. Zakir Hossain et al. “Development of a Bioactive Paper Sensor for Detection of Neurotoxins Using Piezoelectric Inkjet Printing of Sol-Gel-Derived Bioinks,” Analytical Chemistry, vol. 81, pp. 5474-5483. 2009

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References• http://www.sciencedaily.com/videos/2006/1109-

e_coli_handheld_sensor.htm

• http://www.emedicinehealth.com/biological_warfare/article_em.htm

• http://www.cranfield.ac.uk/health/researchareas/biosensorsdiagnostics/p age18795.html

• http://www.guardian.co.uk/world/2008/dec/02/china

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