Lecture 2: Biosensors and Whole Cell Biosensors

Lecture 2: Whole Cell BiosensorsR

Introduction to Biosensors

Definition of Biosensors: Sensors that utilize a bioreceptor for analyte detection. They can be wearable (e.g., watch), integrated into devices (e.g., cell phone), or simple (e.g., paper strip).
Basic Characteristics (brief review from previous lecture):
- Sensors have main components.
- Discussed basic characteristics of a typical biosensor.
Performance Characteristics:
- Selectivity: The ability of a biosensor to detect a target analyte in a complex mixture of chemically similar compounds (e.g., blood). This is crucial for practical applications.
- Sensitivity: High sensitivity allows for earlier and/or less invasive diagnostics.
  - Example: Alzheimer's Biomarkers (Abeta42 peptide): Large quantities of Abeta42 are found in brain plaques, but significantly less in blood, and very minute quantities, if any, in urine. Therefore, a supersensitive assay is required for non-invasive detection.
  - Tear Collection: Tears contain more Aβ peptide than urine or sweat. Schirmer's strip with a specially made collection device is used. After removal, the strip is rolled into the device and centrifuged for $5$ minutes to recover $10\mu L$ of tear fluid.
  - Diagnostic Accuracy (Nature, 2025 & JAMA, 2024):
    - Without advanced blood tests: Dementia specialists achieved $73\%$ accuracy, primary-care physicians only $61\%$ accuracy based on physical exams, cognitive testing, and brain scans.
    - With latest commercial blood tests for Aβ peptide and tau protein (another biomarker): Accuracy improved to $91\%$ . This is, however, for people already exhibiting cognitive symptoms.
  - Limitations of Biomarker Testing in Asymptomatic Individuals (Science, 2024): Many asymptomatic individuals testing positive for Aβ amyloid biomarker may never develop cognitive symptoms. A $65$ -year-old man testing positive has a lifetime risk of Alzheimer's dementia of ~$22\%$, which is only about $1.7$ times higher than the risk for a similar individual testing negative.
- Conclusion on Diagnosis: Alzheimer's and many other diseases should not be diagnosed solely based on a blood test/biosensor. Biosensors are better suited as part of a broader diagnostic process and are very useful for patient management.
- Stability, Reliability, and Reproducibility: These were discussed in class and include self-testing control functions to ensure consistent performance.

Patient Management and Future Applications

Managing Inflammation: Circulating C-reactive protein (CRP) is associated with chronic inflammation and higher mortality risk (Nature BME 2023). Wearable biosensors can measure CRP in sweat (Science, 2024; Nature Rev BME 2025).
Closed-Loop Systems: Used for on-demand drug delivery, such as insulin delivery for diabetics.

Biological Receptors in Biosensors

By definition, anything "biological" can be used.
Most Frequent Types:
- Macromolecules: Proteins (e.g., antibodies), and nucleic acids.
- Whole Cells: Bacteria, plant cells, and animal cells. (Focus of this lecture)

Whole Cell Biosensors: Microorganisms (Bacteria)

Advantages of Bacteria in Sensors:
- Typically more stable than isolated macromolecules (e.g., proteins, receptors).
- Can perform complex multi-step sensing reactions, acting as an "enzymatic microreactor" or "bag of enzymes".
- Can be stabilized and stored for long periods.
- Very inexpensive.
Main Drawbacks:
- Often slow.
- Not very sensitive.
- Reproducibility can be an issue due to natural variation between individual cells.

Bacteria-Based Biosensors for Uranium

Problem: Over $100$ uranium-contaminated sites in the US, with > $6$ trillion liters of waste. Uranyl ion ( $UO_2^{2+}$ ) is the most water-soluble form and spreads easily.
Need for Biosensors: Sensitive detection is crucial for identifying contaminated areas and evaluating bioremediation. While conventional methods (Atomic emission, Mass spectrometry) exist, biosensors can detect bioavailable uranium on-site, not just total uranium, which is of greater interest.
Bacterial Sensing Mechanism:
- Caulobacter sp can thrive in low-nutrient, contaminated environments. It's resistant to uranyl concentrations up to $1mM$ and upregulates several genes in response to uranium.
- Key Gene: urcA is upregulated up to $27$ fold specifically by uranium, not by other heavy metals, indicating selectivity.
Designing the Sensor (Reporter Proteins):
- To make a sensor, a reporter protein is needed to signal urcA gene expression.
- Ideal Reporter: An enzyme catalyzing a detectable, non-toxic product not naturally present in the cell.
- Examples:
  - $\beta$ -Galactosidase (lacZ): A classic reporter. When placed on a plate containing a suitable substrate, its presence leads to a colored (e.g., blue) product upon cleavage of the substrate.
  - Green Fluorescent Protein (GFP): A non-enzymatic reporter from jellyfish. It can be detected by fluorescence without added substrates, making it convenient. It's easily detectable, non-toxic, and not present in other cells or animals.
Biosensor Mechanism:
- The lacZ or GFP gene is placed under the control of the urcA promoter.
- Process: Interaction of $UO_2^{2+}$ with a recognition protein initiates intracellular events, activating a transcription factor (TF X). TF X binds to the urcA promoter, initiating transcription.
- If lacZ/GFP is under this promoter's control, the cell produces the reporter protein when exposed to uranium -> SIGNAL generation.
Performance (Hillson et al., 2007):
- Showed response to $UO_2^{2+}$ with both lacZ (blue product) and GFP (fluorescence) under urcA promoter after $4$ hours of exposure.
- Little/no inhibition from other heavy metals.
- Groundwater samples from contaminated sites could be analyzed, showing clear fluorescent signals under UV light.

Unattended Monitoring and Home Use

Concept: Continuous, real-time environmental monitoring with wireless data transmission to a remote control center (Biosensors & Bioelectronics, 2021).
Advantages: No sample collection/lab analysis, low cost per sample.
Adaptation for Consumers: Biosensing layer applied to a strip, dried for preservation, rehydrated in water sample to detect contaminants (e.g., pesticides).
Challenge: Cost ($).

Water Quality Monitoring (Overall Stress)

Semi-Specific Sensors: Instead of specific contaminants, bacteria can detect overall harmful compounds/conditions by activating stress-responsive promoters (e.g., heat shock, SOS response to DNA damage).
A reporter gene placed under a stress-inducible promoter generates a warning signal when bacteria are exposed to stress-triggering conditions (e.g., DNA/protein damage, toxic chemicals, pH).
Luciferase (lux gene): A superior reporter enzyme that emits light (bioluminescence) from luciferin + ATP + O2.
- Advantage: Rapid response, high sensitivity, stable at elevated temperatures.
- Disadvantage: Requires oxygen and exogenous substrate (for firefly enzyme).
Problem with Stress-Inducible-Only Sensors: If the stress is too severe, bacteria can die, leading to no signal (false negative).
Solution: Self-Testing Function: Combine a stress-inducible reporter with a constitutively expressed reporter.
- Stress-inducible promoter + reporter: Signals hazard.
- Constituent promoter + reporter: Always "on" if bacteria are alive.
- Scenario 1 (Water OK): Constituent reporter is ON, stress-inducible is OFF.
- Scenario 2 (Water NOT OK): Constituent reporter is ON or partially OFF, stress-inducible is ON.
- Scenario 3 (Bacteria Dead): Both reporters are OFF -> Indicates a problem (e.g., high toxicity).
Microtox® Test: A commercial water quality test using bacteria with constitutively expressed lux reporter gene. Used at: Olympic Games (Los Angeles 1984, Atlanta 1996), political events (Democratic convention 2000), Iraqi cities during Gulf War, US Army Corps of Engineers (Pentagon water supply), numerous civilian installations.

Summary of Reporter Genes

Gene/Protein

Pros

Cons

lacZ (\beta $-Gal)</td><td colspan="1" rowspan="1" style="text-align: left;">Wide variety of detection methods (visual by eye)</td><td colspan="1" rowspan="1" style="text-align: left;">Exogenous substrate required</td></tr><tr><td colspan="1" rowspan="1" style="text-align: left;"><code>gfp</code> (green fluorescent protein)</td><td colspan="1" rowspan="1" style="text-align: left;">No substrate requirement for fluorescence</td><td colspan="1" rowspan="1" style="text-align: left;">Lower sensitivity, slower response</td></tr><tr><td colspan="1" rowspan="1" style="text-align: left;"><code>luc</code> (firefly luciferase)</td><td colspan="1" rowspan="1" style="text-align: left;">Rapid response; high sensitivity, stable at elevated temperatures</td><td colspan="1" rowspan="1" style="text-align: left;">Requirements for oxygen and exogenous substrate (firefly enzyme)</td></tr></tbody></table><h5 id="69d5f05f-e46c-4a4a-a074-4d206d6798ea" data-toc-id="69d5f05f-e46c-4a4a-a074-4d206d6798ea" collapsed="false" seolevelmigrated="true">Comparison of Uranium Biosensors</h5><ul><li>Bacterial Biosensor (earlier discussed):<ul><li>Disadvantages: Lower selectivity compared to chemical methods, takes$ 3-4 $hours for results (vs.$ 10-15 $minutes for conventional analysis), lower sensitivity (not at pmol level).</li></ul></li><li>DNAzyme Biosensor (PNAS, 2007): A small catalytic DNA that cleaves itself only in the presence of$ UO2^{2+} $. Binding of$ UO2^{2+} $leads to cleavage and release of a fluorophore.<ul><li>Detection Limit:$ 11 $parts per trillion (~$ 45pM).

Selectivity: >1 $-million-fold over many other metal ions.</li><li>Detection Time:$ 4-5 $minutes.</li><li>Conclusion: This biosensor is as good as most modern analytical instruments.</li></ul></li></ul><h5 id="c0059294-517a-4866-bab4-7b60f03f6395" data-toc-id="c0059294-517a-4866-bab4-7b60f03f6395" collapsed="false" seolevelmigrated="true">Improving Reliability and Speed</h5><ul><li>Problem: Living systems inherently have variations (reporter gene expression, cell sensitivity, growth conditions, etc.), leading to large error bars in responses.</li><li>Solution: Cell-Free Systems (in vitro transcription - IVT):<ul><li>Eliminates the need for cells, reducing variability.</li><li>Uses fluorescent RNA-level output instead of protein, making it faster.</li><li>Mechanism: Analyte binding to a transcription factor results in RNA transcription, which then has a fluorescent label.</li><li>Example (Nature Biotech, 2020): Biosensing$ Zn^{2+} $and other metals in municipal water. The system can be freeze-dried for field deployment and activated on rehydration. Limit of Detection (LoD) is below EPA guidelines for$ Zn^{2+} $.</li><li>Benefit: Faster, but still a balance of speed and complexity.</li></ul></li></ul><h4 id="9d1480b7-40f1-42fb-9dab-6b59a89db990" data-toc-id="9d1480b7-40f1-42fb-9dab-6b59a89db990" collapsed="false" seolevelmigrated="true">Pharma Applications</h4><ul><li>Ames Test (Standard Mutagenicity Assay):<ul><li>Uses Salmonella strains unable to synthesize histidine (<code>his-</code>). These strains cannot grow without histidine.</li><li>Reversion Assay: Bacteria are spread on agar with minimal histidine. They grow but then die when His is consumed, unless a mutation restores <code>his</code> gene function, allowing them to produce His and continue growing.</li><li>Mutagen Detection: Mutagenic substances increase the rate of these "reversions," leading to more colonies, often in a dose-dependent manner.</li></ul></li><li>Bioluminescent Salmonella for Mutagenicity (Mutagenesis, 2007 - Pfizer):<ul><li>Principle: Introduce the <code>lux</code> gene into histidine-dependent Salmonella.</li><li>In the absence of histidine, <code>his-</code> cells cannot sustain bioluminescence (due to insufficient FMNH2 for luciferase), while revertant (<code>his+</code>) cells continue to emit light.</li><li>Revertant colonies are visualized and quantified using a photon-counting camera.</li><li>Advantage: Provides an economical, high-throughput method for early mutagenicity assessment of drug candidates, reducing the ~$ 12\% $of candidates dropped due to genetic safety concerns.</li></ul></li></ul><h5 id="06847329-111b-4eae-a6b3-95bfff27547a" data-toc-id="06847329-111b-4eae-a6b3-95bfff27547a" collapsed="false" seolevelmigrated="true">Medical Diagnostics and Therapeutics</h5><ul><li>Bacteria for Liver Metastasis Detection (E.coli probiotic, Science Translation Med, 2015):<ul><li>Engineered <code>E.coli</code> with <code>LacZ</code> administered intravenously.</li><li>Bacteria specifically amplify within metastatic tumors (systemically injected) in the liver, but not in other tissues.</li><li>Signal (from <code>LacZ</code> reporter) can be detected in urine after substrate administration, allowing early, non-invasive detection of liver metastasis.</li></ul></li><li>Biosensor for Liver Disease (Nature Com, 2021):<ul><li>Liver disease causes ~$ 2 $million deaths/year; current diagnostics are hospital-based.</li><li>Engineered bacteria detect bile salts (a biomarker of liver dysfunction).</li><li>Mechanism: Ligand-induced dimerization of a bile salt receptor (LBD) triggers signal generation (e.g., using CPRG, hydrolyzed to chlorophenol red + Gal).</li></ul></li><li>Phenylketonuria (PKU) Treatment (Synlogic, Nature Com, 2020; Nature Metabolism, 2023):<ul><li>PKU: Genetic disease with reduced phenylalanine hydroxylase, leading to elevated plasma phenylalanine (Phe) and cognitive impairment.</li><li>Engineered Probiotic (E.coli): Converts Phe to trans-cinnamic acid in the gut; this is then converted to hippuric acid in the liver and excreted in urine.</li><li>Mechanism: Oral bacteria remain inactive until reaching the gut, where an internal "switch" triggers enzyme production based on the environment.</li><li>Safety: No long-term colonization in healthy humans observed.</li><li>Outcome: Completed Phase$ 2 $trials in 2022, but failed pivotal Phase$ 3 $in 2024.</li></ul></li></ul><h4 id="1a495d20-761b-494b-8652-69496c270542" data-toc-id="1a495d20-761b-494b-8652-69496c270542" collapsed="false" seolevelmigrated="true">Whole Cell Biosensors: Plant Cells</h4><ul><li>General Assessment: Rarely used in biosensors.</li><li>Disadvantages: Expensive, often slow (due to cell walls), and useful only for very specialized applications.</li><li>Potential "Advantages": Plants can "feed" themselves.</li><li>Example: Potentiometric Biosensor for Pathogenic Microorganisms (TRENDS in Plant Science, 2002; Bioelectrochemistry, 2007):<ul><li>Plants detect pathogens via general elicitors (molecules characteristic of a class of microorganisms), such as flagellin (from bacterial flagella).</li><li>Receptor: Plants recognize flagellin using the extracellular receptor FLS2.</li><li>Signal: Flagellin binding to FLS2 elicits a cellular response, leading to an electrical signal (movement of ions/electrical response).</li><li>Application: A plant cell-based sensor with an electrode transducer gives an electrical readout upon flagellin binding (e.g., great response at$ 100nM $flagellin).</li><li>Critical Evaluation: Unlikely to be a useful biosensor as similar electrical responses might occur from non-specific stimuli (e.g., pH change). Using antibodies could be a more effective alternative for flagellin detection.</li></ul></li><li>Interesting Concept: Bioluminescent houseplants for home health monitoring (Science 2018, Nature 2024, Light.bio).<ul><li>A new product (bioluminescent houseplant) hit the US market in 2024 for$ 34.99 $and sold out quickly.</li></ul></li></ul><h4 id="72603050-a3d6-46d6-b1f3-409a8c364080" data-toc-id="72603050-a3d6-46d6-b1f3-409a8c364080" collapsed="false" seolevelmigrated="true">Whole Cell Biosensors: Animal Cells</h4><ul><li>Main Drawback: Expensive.</li><li>Advantages: Offer unique possibilities in biomedical analysis, sometimes the only available option.</li><li>Usage: Used in specialized sensors as individual cells or tissue slices.</li><li>Early Applications:<ul><li>Chemical Warfare Agents: Detection of DFP (diisopropylfluorophosphate,$ 25\mu M $) inhibiting spontaneous firing of spinal cord neurons; effects of VX and GD (soman) on rhythmic firing, measured with glass microelectrodes. These show specific or semi-specific responses.</li></ul></li><li>Recent Applications:<ul><li>Water Quality Monitoring (Fish Gill Cells):<ul><li>Problem: Industrial chemicals, fertilizers, and pesticides pollute water globally (Current Opinion in Biotechnology, 2017). Traditional fish acute toxicity tests are labor and animal-intensive ($ 42-60 $fish exposed for$ 96 $hours,$ 3 $million fish used in North America in 2011).</li><li>Solution: In vitro cell-based assay using fish gill cell line (RTgill-W1) on a chip with microelectrodes.</li><li>Performance (Environ. Sci. Technol., 2013): Excellent correlation for$ 75\% $of tested compounds with traditional fish assays.</li><li>US Army Device (JOVE, 2016): A field-portable device for drinking water contamination meeting specifications: broad spectrum detection at relevant concentrations, rapid detection (max$ 1 $hour),$ 9 $months shelf-life for biological components.</li></ul></li><li>Cardiomyocyte-Based Biosensor (hESC-derived, Biosensors and Bioelectronics, 2013):<ul><li>Cardiomyocytes (e.g., derived from human Embryonic Stem Cells - hESC) are placed on an electrode.</li><li>Records frequency of their contraction.</li><li>Application: For evaluating anti-arrhythmic drugs (e.g., Verapamil, a Ca-channel blocker), by observing changes in contraction frequency.</li></ul></li></ul></li></ul><h5 id="93c55f76-0c1b-4914-ab21-f13c4ae38555" data-toc-id="93c55f76-0c1b-4914-ab21-f13c4ae38555" collapsed="false" seolevelmigrated="true">Hijacking Cells' Signaling: G Protein-Coupled Receptors (GPCRs)</h5><ul><li>GPCRs (Science, 2007): Large family of transmembrane receptors (seven transmembrane domains) that sense extracellular signals (hormones, neurotransmitters, light, odors).<ul><li>Upon activation, GPCRs initiate intracellular signal transduction cascades, leading to cellular responses.</li></ul></li><li>Signaling via G-proteins (Science, 2002):<ul><li>GPCRs are associated with inactive G-proteins (with$ \alpha $,$ \beta $,$ \gamma $subunits and bound GDP).</li><li>Ligand binding to GPCR causes conformational change, activating the G-protein via replacement of GDP with GTP.</li><li>GTP/GDP exchange causes dissociation of G-protein subunits, which then activate effector proteins/enzymes, amplifying the signal inside the cell (producing "second messengers" like cAMP, cGMP, Ca$ ^{2+} $).</li><li>G-proteins unify and integrate intracellular signaling, much like "cells' own biosensors."</li></ul></li><li>Whole-Cell G-protein Sensor (J. Biol. Chem., 2007):<ul><li>Concept: Introduce two fluorescent labels (e.g., YFP and CFP) into the G-protein.</li><li>Mechanism: Upon G-protein activation and conformational change, the labels move apart. This alters the FRET (Förster resonance energy transfer) signal, increasing the "cyan" signal and decreasing the "yellow" signal. The ratio provides a very nice response.</li><li>Application (Morphine Receptor Biosensor): Monitoring morphine activation in living cells. The labels do not affect the biochemical events.</li></ul></li><li>Study of Morphine Analogs (J. Biol. Chem., 2007):<ul><li>G-protein activation can be used to investigate pharmacological properties of compounds acting on GPCRs, serving as a powerful tool for drug discovery (approx.$ 30\% $of current drugs target GPCRs).</li><li>Allows comparison of relative potencies (e.g., morphine vs. morphinone) and determination of IC$ _{50}$$.

Cannabinoid Receptor Biosensors (CB1/CB2, Nature Com, 2022):

CB1 and CB2 receptors are also GPCRs. Biosensors for cannabinoids and synthetic analogs are useful for drug development (e.g., targeting CB2 for therapeutic effects while avoiding psychotropic CB1 effects), screening illicit drugs, and quality control of cannabis potency.
Yeast System: Yeast is an excellent host because its pheromone pathway is similar to mammalian GPCR pathways.
Concept: Hijack yeast pheromone pathway by replacing the pheromone receptor with cannabinoid receptors (CB2) and monitor downstream response with a reporter (e.g., luciferase).
Application: Calibration for THC in body fluids, portable versions developed.

Specialized Animal Cell Biosensors

Red Blood Cell (RBC) Biosensor (ACS Synth. Biol., 2024):
- Unique Feature: RBCs have a very long circulation time and reside in blood, making them ideal sentinel cells for in situ sensing.
- Challenge: RBCs lose their nucleus during maturation, limiting common genetic engineering approaches.
- Concept: Binding of an extracellular ligand leads to reconstitution of an intracellular split-output protein that generates light (optical signal), e.g., luciferase, for non-invasive in vivo detection.
Allergy Profiling Sensor (Nature Com, 2014):
- Mechanism of Allergy: Allergen-bound IgE triggers mast cell/basophil activation, releasing histamine, which activates histamine receptors (H1-4, also GPCRs) and causes allergic symptoms.
- Designer Cell: Mammalian cells engineered with a signaling cascade that couples histamine input to the production of a reporter (e.g., SEAP - secreted Alkaline Phosphatase, or YFP - Yellow Fluorescent Protein).
- Application: Exposure of human serum to these cells mimics patient-specific in vivo allergic conditions. The reporter signal (fluorescence of citrine for YFP, or SEAP signal) provides sensitive and selective personalized allergy profiles in real-time (e.g., allergic to Hazel but not Bermuda grass), correlating well with lab tests.

Bioreceptors: Comparison Summary

Bioreceptor	Advantage	Disadvantage
Macromolecules (proteins, DNA)	Sensitive, no side reactions	Need to be identified, isolated, or designed
Microorganisms	Multi-step reactions are possible, cheap	Low selectivity and sensitivity, slow
Eukaryotic cells (mammalian)	Enable unique assays	Relatively expensive, unstable
Tissue slices	Minimal preparation; cheap	Slow diffusion/side reactions
Plant cells	Only useful in some very special cases	Expensive (if cells), slow

Basic Assay Systems

Flow-through System: Analyte is injected into a continuous flow over the sensor. Output signal is plotted against time.
Closed System: Analyte is added to a closed container with the sensor. Signal changes over time, sometimes without wash steps.

Typical Response Characteristics

Signal vs. Analyte Concentration: A typical response curve shows signal increasing with analyte concentration until bioreceptor saturation.
Limit of Detection (LOD): The minimum detectable concentration of the analyte in a sample. Often significantly reduced in real samples compared to model solutions due to competing substances, inhibitors, etc.
Upper Limit: The point at which the bioreceptor is saturated and can no longer detect higher concentrations of the analyte.
Dynamic Range: The range of analyte concentrations over which the sensor provides a reliable and quantifiable signal.
Sensitivity: The slope of the response curve within the dynamic range, indicating how much the signal changes for a given change in analyte concentration.