Lecture 2: Biosensors and Whole Cell Biosensors

Flashcards for Lecture 2 covering biosensors, their characteristics, various types of whole-cell biosensors, and specific applications, including reporter proteins and cell signaling mechanisms.

How are biosensors defined?

Biosensors are sensors that use a bioreceptor for analyte detection.

What are the main components mentioned for a typical biosensor?

A bioreceptor for analyte detection, and other sensor components (e.g., transducer, detector).

What does biosensor selectivity refer to in practice?

Biosensors are most useful for analyte detection in a complex mixture of chemically similar compounds.

What does high sensitivity in biosensors enable?

Earlier and/or less invasive diagnostics.

What is a key challenge in detecting Alzheimer's biomarkers like Abeta42 in blood or urine?

Minute quantities are present, requiring a supersensitive assay.

Besides selectivity and sensitivity, what other performance characteristics are crucial for biosensors?

Stability, reliability, and reproducibility.

Where can Aβ peptide be found in higher concentrations than urine or sweat for non-invasive detection?

Tears.

How were tears collected for Aβ peptide analysis in the study mentioned?

Using a Schirmer's strip with a specially made collection device, followed by centrifugation.

What was the diagnostic accuracy of dementia specialists relying solely on physical exams, cognitive testing, and brain scans for Alzheimer's?

73%.

What was the diagnostic accuracy when using one of the latest commercial blood tests for Aβ peptide and tau protein?

91%.

What is a concern when diagnosing asymptomatic people using a positive Aβ amyloid biomarker test?

Many would never develop cognitive symptoms, and a positive test only moderately increases lifetime risk.

What is the recommended role of biosensors in diagnosing complex diseases like Alzheimer's?

They are useful as part of the diagnostic process, not as the sole basis for diagnosis.

What is circulating CRP associated with?

Chronic inflammation and higher mortality risk.

What four broad categories of biological receptors can be used in biosensors?

Antibodies, nucleic acids, macromolecules (proteins, nucleic acids), and whole cells (bacteria, plant, and animal cells).

What are two main advantages of using bacteria in sensors?

They are typically more stable than isolated macromolecules and can perform complex multi-step sensing reactions.

What are two main drawbacks of bacteria-based biosensors?

They are often slow, not very sensitive, and reproducibility can be a problem.

Why are bacteria-based biosensors useful for uranium detection, beyond detecting total uranium?

They can detect bioavailable uranium on-site.

Which bacterium is known to live in uranium-contaminated environments and sense uranium?

Caulobacter sp.

What specific gene in Caulobacter sp is significantly upregulated in response to uranium and is selective?

urcA.

To make a sensor from the urcA gene expression, what is needed?

A reporter protein that generates a detectable signal.

What is a classic enzymatic reporter used in combination with substrates that produce colored products?

β-Galactosidase (lacZ).

What is a disadvantage of lacZ as a reporter for some biosensors?

An exogenous chromogenic substrate needs to be added for detection.

What is an example of a non-enzymatic reporter protein that generates a detectable signal without added substrates?

Green fluorescent protein (GFP).

How is a bacterium engineered to create a uranium biosensor using a reporter gene?

The lacZ or GFP gene is placed under the control of the urcA promoter.

What is observed when a bacterium engineered with lacZ under the urcA promoter is exposed to UO2 2+?

The bacterium expresses lacZ, and its presence is detected as a blue-colored product.

What benefit does unattended monitoring with biosensors offer in environmental monitoring?

No need to collect samples and send them to the lab, and wireless data transmission.

How do bacteria-based biosensors indicate overall water quality by responding to harmful compounds?

By placing a reporter gene under the control of a stress-responsive promoter.

What happens when a reporter gene under a stress-responsive promoter detects stress-triggering conditions?

The bacteria generate a warning signal.

What is an effective light-emitting enzyme used as a reporter for bioluminescence?

Luciferase.

What is a significant problem with a simple stress-inducible biosensor setup?

If the stress is too severe, the bacteria will die, leading to no warning signal.

How can the problem of bacteria dying in a stress-inducible biosensor be controlled?

By combining it with a constitutively expressed reporter gene.

What function does a constitutively expressed reporter provide in a biosensor?

A 'self-testing' function to confirm the bacteria are alive and the sensor is working properly.

Name a commercial test that uses bacteria constitutively expressing the lux reporter gene to monitor water quality.

Microtox®.

Briefly state a pro and con for lacZ (β-Gal) as a reporter.

Pro: Wide variety of detection methods; Con: Exogenous substrate is required.

Briefly state a pro and con for GFP as a reporter.

Pro: No substrate requirement for fluorescence; Con: Lower sensitivity, slower response.

Briefly state a pro and con for luc (firefly luciferase) as a reporter.

Pro: Rapid response, high sensitivity; Con: Requires oxygen and exogenous substrate.

What were two drawbacks of the initial uranium biosensor (bacterium-based)?

Selectivity was not great compared to chemical methods, and it took 3-4 hours for results.

What advanced type of biosensor was described for uranium detection that rivaled modern analytical instruments?

A small catalytic DNA (DNAzyme) that cleaves itself in the presence of UO2 2+.

Why do living systems often exhibit large variations in biosensor response (error bars)?

Different levels of reporter gene expression, cell sensitivity variations, and subtle growth condition changes.

What method can improve reliability and accuracy by eliminating the need for cells?

A cell-free system using in-vitro transcription (IVT).

What is the primary purpose of the Ames test?

To test new chemicals and drugs for mutagenicity.

How is the Ames test performed?

It uses Salmonella strains unable to synthesize histidine, and measures their reversion to the ability to grow without histidine in the presence of a test substance.

How can a bioluminescent Salmonella be used in mutagenicity assessment?

By introducing the lux gene, revertant colonies can be detected by emitting light.

What is an example of an engineered E. coli probiotic biosensor used for early detection in diagnostics, as discussed?

A biosensor for early detection of liver metastasis.

How did the engineered E. coli biosensor detect liver metastasis in mice?

It selectively amplified within metastatic tumors and produced a signal detectable in urine.

What is a biomarker of liver dysfunction that can be detected by engineered bacteria, as discussed?

Bile salts.

What genetic disease does Synlogic's engineered E.coli target by converting phenylalanine to other metabolites?

Phenylketonuria (PKU).

Why are plant cells generally not preferred for biosensors compared to other cell types?

They are expensive if using whole cells, often slow due to cell walls, and only useful for specialized applications.

What are GPCRs (G protein-coupled receptors)?

A large family of transmembrane protein receptors that sense extracellular signals (e.g., hormones, neurotransmitters) and initiate intracellular signal transduction.

What is the Limit of Detection (LOD) in a biosensor?

The minimum detectable concentration of the analyte in a sample.