Biosensor technology Flashcards
What is the difference between data and information?
Data can be raw figures and facts that are not put in a context, while information is data that is structured and put in a context
What is the definition of a biosensor?
A self-contained integrated device that can provide specific quantitative analytical data and uses a biorecognition element
What are the components of a biosensor?
Biorecognition element: enables specific recognition of the analyte
Interfacial component: facilitates immobilization of the bioreceptor and prevents unspecific binding of analyte
Transducer: converts the recognition event into a measurable signal
List some biorecognition elements
- Antibodies
- Enzymes
- DNA
- Aptamers
- Peptides
- Organelles
- Cells
List some transducer technologies
- Surface Plasmon Resonance
- Fluorescence
- Colorimetric
- Amperometric
- Potentiometric
- Piezoelectric
- Calorimetric
What are some differences between a biosensor and a bioassay?
An assay is an analytic procedure for quantitatively assessing the presence, amount or activity of a target. Uses BRE and is never an isolated process
What are some advantages of a biosensor compared to a clinical lab?
From a biosensor you can get real-time information and point of care. It is easier to obtain multi-time-point data.
What is a biorecognition element (BRE)?
Interactions between the recognition element and the analyte
Why does affinity matter for a affinity-based BRE?
A higher affinity, which means lower dissociation constant, of the bioregocnition for the analyte results in a larger sensor signal for a given analyte conc.
How does the turnover (kcat) affect the sensor signal?
The turnover is the maximum number of chemical conversions of substrate per second.
A higher kcat results in more product and therefore a larger sensor signal
What is unspecific binding?
It is binding of anything else than the analyte to the sensor or binding of the analyte to other parts of the signal generating components of the transducer.
What do you have to consider about the sample that you will use?
You must take the sample matrix and the analyte into consideration. Some properties that you have to consider are:
- Analyte size
- Chemical properties of analyte
- Range of analyte conc
- Which specificity and selectivity is required
- Complexity of the sample
What is the specificity of a sensor what is important to. consider?
The specificity of a sensor is the ability to explicitly assess the analyte in the presence of other components. The affinity for the analyte must be much higher than for binding other molecules that can bind to the BRE.
For detection of multiple different analyte we need multiple BRE for the specific analytes
What is selectivity?
A sensor array is selective if it can discriminate between different substances in the sample
What is the limit-of-detection (LOD)?
The lowest conc. of a substance that can be distinguished from the absence of that substance within a stated confidence limit.
Y(LOD) = y(blank) + k*σ(blank)
k is usually 3
What is the limit-of-quantification (LOQ)?
More reliable analytical results are obtained when the conc. is above a higher limit, LOQ.
Y(LOQ) = y(blank) + 10*σ(blank)
What is the dynamic range of the sensor?
The ratio between the upper and lower detection limits. The lower limit is the LOD/LOQ and the upper limit is where the response deviates from the assumed calibration function, due to saturation of BRE.
How can you decrease the LOD/LOQ?
Use a BRE with higher affinity for the analyte. For an enzymatic BRE you can use a BRE with higher turnover (kcat). You can also reduce noise-lever of the sensor, so you decrease the 3*σ
How do you prevent saturation of BRE?
Use BRE with lower affinity for the analyte (large KD) or dilute the sample or increase the sensor surface area
What is the sensitivity of a sensor?
The slope of the linear region.
The steeper slope the higher the sensitivity is. This means that there is a better possibility to distinguish small changes in the analyte conc = resolution.
The resolution is the smallest detectable change in analyte conc.
What is sensor noise and what can cause it?
The noise is random variation of sensor output that are unrelated to the sensor input. The noise can arise from random processes in the sensor function from electronic noise in the readout equipment.
It is important to look at the signal-to-noise ratio. A high sensitivity and low noise level gives a high resolution
What is accuracy, precision and reproducibility?
Accuracy: closeness of the measurements to a specific value
Precision: closeness of the measurements to each other
Reproducibility: ability if the sensor to produce identical results whenever the same sample is measured more than once
How can one improve the reproducibility?
The main reason for poor reproducibility is manual handling. So minimize manual handling.
Factors that affect the sensor robustness are environmental changes (temp and storage conditions) and sample interference
What are the different transducer categories and what do they measure?
- Electrochemical: detects redox reactions. Enzymatic reaction that generates electrochemical active species
- Optical: detects changes in wavelength or intensity. Analyte that are either colored or fluorescent or labeled or analyte-binding induces a change in refractive index.
- Gravimetric: detects changes in mass or viscoelastic properties. The change in the resonance frequency of an oscillating senor surface when the mass changes upon analyte binding
How can one use gold nanoparticles as a transducer?
As gold nanoparticles aggrigate they change color - colorimetric detection.
They can absorb and scatter light very efficiently and are very stable (non-oxidizing) and they can be used to in gold-thiol-chemistry for functionalization for conjugation of AB
How can we optimize the naked eye detection of color change?
- The color change should appear within a narrow spectral range
- Produce a distint and sharp color transition
- Appear in the green ange where our eyes are the most sensitive
Describe the DNA hybridization triggered aggrigation using gold nanoparticles.
When the target DNA is present in the sample, it hybridizes with the probe on the gold nanoparticle surface, causing the nanoparticles to aggregate.
The melting temp is proportional to the number of interactions involved. More interactions result in a higher melting temp.
What are cantilever biosensors?
It is a microfabricated beam clamped at one and and at the other and, the surface is functionalized with bioreceptors (often affinity binding).
Deflection of the cantilever is caused by repulsive forces between bound analytes
What is a piezoelectric transducer?
The piezoelectric effect is the ability of a material to produce a voltage when mechanically stressed vice vera.
Used in static gravimetric sensor to transduce an analyte-induced bending of the cantilever load into an electric response
Used in dynamic gravimetric sensors to induce oscillations of the sensor surface (the resonance freq changes upon analyte binding)
What is a bulk acoustic wave (BAW) biosensor?
A type of biosensor that utilizes a thin piezoelectric material to detect changes in mass, viscosity, or elasticity caused by biomolecular interactions.
How does QCM-D work?
The driving voltage is intermittently switched off and the decay is monitored. Form the decay curve, the resonance freq and the energy dissipation are extracted. The disspation gives info about the energy loss in the system - how soft or viscoelastic the layer is
Why use enzyme-based recognition?
Possible to measure reaction products: pH, electrons, heat, and light
What is an allosteric enzyme?
An effector modulates the catalytic rate by inducing conformational change in protein structure -> activating or deactivting
Why use antibodies as BRE?
Perfect fit in terms of structure and chemical complementary towards the epitope of the antigen
What are aptamers?
Synthetic DNA/RNA receptors that exploits the ability of DNA and RNA to selectively recognize and bind to proteins or other molecules
Describe the SELEX
- Starts with library with random sequences
- Library is mixed with target
- Unbound sequences are removed
- Bound sequences are eluted and amplified using PCR
- Repeated 10-15X to narrow down the number of sequences and extract those with highest affinity
What are some advantages of using aptamers?
Adv:
- inexpensive and fast selection
- Uniform activity regardless of batch
- Affinity parameters can be controlled
- Unlimited shelf life
- Recover original conf. efter thermal shock
- Can diversify properties and function
What are some disadvantages of using antibodies?
- Screening monoclonal ab is expensive and time consuming
- Activity varies from batch to batch
- Difficult to modify
- Limited shelf life
- Temp causes irriversable denaturation
What are aptanzymes?
RNAzymes or DNAzymes have the potential to replace enzymes as BRE. Share the same properties as aptamers
What are molecular beacon aptamers?
The binding to analyte induces conformational changes that can open hairpin and “turn on” quenched fluorophore
