Exam 2 Flashcards
What physiological parameters can a pulse oximeter measure?
oxygen saturation in blood (SpO2) and heart rate (pulse)
Define SpO2
- The ratio of oxygenated over deoxygenated blood (oxygenated/deoxygenated)
- SpO2 is the concentration of oxygenated blood, specifically the amount of oxygen carrying hemoglobin in blood
Identify two wavelengths from these spectra that a pulse oximeter uses.
Pulse oximeter uses these as 660nm corresponds to red light absorbance and 940nm to NIR absorbance, the ratio of these two absorbances indicate SpO2
A miniature spectrophotometer can obtain a whole spectrum instantaneously. Graphically illustrate how it works.
Spectrometry is achieved by mechanically rotating and/or moving a prism and a mirror such that the wavelength λ of the light passing through a cuvette can be gradually changed against time
Using the given refractive indices of core and cladding, calculate the critical angle (θC)
θC = arcsin(Ncladding/Ncore)
Using the given θC, calculate the numerical aperture (NA).
NA = sin(x)
How can a reflection probe deliver the excitation light AND collect the scattered light?
Light source is delivered through the core and material/system reflects/backscatters this. Light is transferred to shell side bundle of fibers then to mini spectrometer.
Calculate absorbance
A = log(Io/I)
Why is a fluorescent dye superior to colorimetric dyes?
Fluorescence photometry has a greater sensitivity than normal absorption photometry in terms of detection. Can detect trace amounts of dye.
Sort the fluorescent dyes in the ascending order of excitation wavelength (or emission wavelength) through examining their chemical structures.
DAPI
- Excitation: 360 (purple)
- Emission: 460 (blue)
Fluorescein
- Excitation: 490 (blue)
- Emission:520 (green)
Rhodamine B
- Excitation: 540 (green)
- Emission: 580 (yellow/red)
Cy3
- Excitation: 550 (green)
- Emission: 650 (orange)
Cy5
- Excitation: 570 (red)
- Emission: 670 (red)
What is DNA intercalating dye? How does it work?
Dye that doesn’t require pre-assay conjugation to a target molecule or bioreceptor (ie antibody) before assay
GFP: protein that emits green when excited with blue light
- Used as marker for gene expression by inserting gene for GFP when inserting
target gene into organism so if gene inserted successfully then GFP synthesized
and green fluorescence observed
SYBR Family
- Cyanine dye (Cy3, Cy5)
- Binds to dsDNA and exhibits strong green fluorescence = intercalating.
- Quantifies dsDNA
- Functions as both dye and bioreceptor
Ethium Bromide
- Also intercalating with dsDNA
- Functions as both dye and bioreceptor
Explain photobleaching. How can you minimize photobleaching of fluorescent dyes?
When fluorescent dye are exposed to light continuously = leads to destruction and fading of fluorescence over time
- To overcome = quantum dots. very fine semiconductor nanocrystals that absorb UV light (excitation) and emit visible light = artificial fluorescence
Describe the fluorescence of NAD+ vs. NADH. Repeat this question for FAD+, FADH, FADH2.
NAD+ (oxidized)
NADH (reduced)
- Both absorb deep UV
- NADH emits blue
FAD+ (Fully oxidized)
FADH (partially reduced)
FADH2 (Fully reduced)
- All absorb blue light
- Only FAD+ emits green
Which component in urine is responsible for autofluorescence? What are their excitation and emission colors?
Urobilin = molecule in urine responsible for yellow coloration and green fluorescence of urine.
- Excitation: blue
- Emission: green
Plant leaves are green because of chlorophyll. Among RGB, identify the colors that chlorophyll absorbs. Chlorophyll also exhibit fluorescence. Identify its excitation and emission colors.
Plants absorb red and blue wavelengths to generate green coloration
- Excitation: blue/red
- Emission: red
How can you isolate just the fluorescence emission from a sample?
Dichroic mirror: reflects excitation light and allows emission light to pass through. Optical fibers: Low pass, high pass, band pass filters. Light source like laser or LED
Emission light detected at any OTHER angle than 0°
Most common angles used are 180° (back scatter) or 90° (side scatter)
Sketch the light intensity (not absorbance) spectra of fluorescein with the reflection probe 1-cm above the air-water interface and 1-cm below the interface.
Above: Expect to see the excitation reflection of fluorescein around 490 and so a weaker emission wavelength caught around 520.
Below: Expect to see little to no reflection of excitation reflection and only really the
emission around 520
Using the standard electrode potentials, identify E° for the given electrochemical cell. In addition, using this E° and given concentrations of electrolytes, calculate E.
E = E0 + (0.059/# electrons used)log(M1+/M2+)
Why is a calomel electrode preferred over a hydrogen electrode as a reference electrode?
Hydrogen electrode is difficult to use
Calomel electrodes have much simpler redox reaction.
Define conductivity and resistivity
Conductivity: Used as measure in conductometric biosensors
- inverse of restivity
Resistivity: measure of resisting power of a specified material to the flow of an electric current.
- Inverse of conductivity
- (RA/l), l = length of specimen
Why do you need TL082 over LM741 in measuring pH?
H electrode has very high resistance = generates low current so op-amp with very high input impedance is needed
Create an op-amp circuit that will generate +4 V at pH 4, +7 V at pH 7, etc.
- Change gain stage to be NON-INVERTING
You can use a pH meter in lieu of an op-amp circuit for a fluoride ion-selective electrode. Briefly explain how this is possible. What is the major difference in their standard curves?
- fluoride ions are negatively charged, so as the concentration of anions increased there was a respective increase in pH due to the fact that more negative ions meant less positive charge (hydrogen ions) in the solution (so the pH probe read that there was less hydrogen ions in solution)
- pH standard curve: as hydrogen ion concentration increased, pH meter reading decreased (inverse relationship)
- Fluoride standard curve: as fluoride ion concentration increased, pH meter reading increased (direct relationship)
GDH-PQQ is the de facto standard in current glucose sensing. Identify its two major benefits over the other methods.
- GDH does not require oxygen
- PQQ has rapid electron transfer rate (quicker sensing)
What is continuous glucose monitoring (CGM)?
- Sometimes need to monitor blood-glucose levels throughout the entire day rather than
just at a few points of the day - Electrode needs to be inserted into body (this enables measurements in vitro) with
glucose levels measured every 1 to 5 min
Why are your commercial glucose meter readings higher than the actual glucose concentrations?
Glucose meters often are corrected to show “plasma” equivalent concentrations instead of the actual glucose concentrations in “whole blood.”
LFA
A) Signal transduction
B) Detection method
C) How samples introduced
D) How unbound molecules are removed
A) Gold nanoparticles
B) Colorimetric (optical)
C) Dipping (capillary action)
D) Spontaneous washing
- Green and some blue absorbed results in pink coloration
- Cheaper than ELISA
ELISA
A) Signal transduction
B) Detection method
C) How samples introduced
D) How unbound molecules are removed
A) Enzyme substrate
B) Colorimetric (optical) / electrochemical
C) Pipetting
D) Pipetting
ELISA is typically quantified by the colorimetry of the enzyme-substrate pair. Can it be replaced with something else?
Fluorescent detection and Electrochemical detection: read current changes from each well
Compare polyclonal and monoclonal antibodies.
Polyclonal: a mixture of antibodies that recognize different portions (epitopes) of an antigen
Monoclonal: an antibody that recognizes only one type of epitope
What is aptamer?
Artificial nucleic acids (DNA or RNA) that recognize or bind to specific antigens, just like antibodies. Aptamers are mostly single-chain nucleic acids, either single-stranded DNA (ssDNA) or RNA
Describe the ELONA procedure.
Same process as ELISA, antigen sandwiched between two aptamers (aptasensor)
Washing steps in ELISA can be simplified by using a reflection probe or a microchannel.
Briefly explain those two methods.
Reflection probe:
- Have antibodies immobilized onto surface if exposed optical fiber and passivating proteins too, then dip into target solution followed by rinse solution then dipped in secondary antibody and antibody-to-antibody conjugation that has fluorescent dye conjugated
Microchannel:
- Target solution continuously flowing through microchannel with antibodies immobilized onto inner surface at specific location of microchannel -> no rinsing as continuous flow does this
- Light delivered to location where immobilization is and a light sensor reads signal on other side
Describe how interdigitated microelectrode (IME) works for detecting large-sized biological targets, such as bacteria.
- Doesn’t require secondary antibody, enzyme-substrate pair, fluorescent dye
- Antibodies are immobilized on surface where 2 electrodes patterened in configuration =interdigitated array
- Big target like bacteria binds to surface bound antibodies to make bridge between
elctrodes and lower resistance - If made smaller then can bind smaller targets like viruses and proteins
If the coloration of ELISA plate is YELLOW, which color channel (among RGB) should you use in evaluating absorbance?
Blue because is what absorbs most so then have better correlation between a color change and absorbance value
If the coloration of ELISA plate is PINK, which color channel (among RGB) should you use in evaluating absorbance?
Green
Describe photolithography
- Etches a trench into light sensitive film
- Put glass cover over to make channels
Describe soft lithography
- Same technique as photolithography
- Pour polymer gel into mold and then bond another layer of polymer gel to create top
Why is soft lithography more popular?
Cheaper and has mass production qualities
What is electroosmotic flow (EOF)?
- In capillary electrophoresis: high voltage applied at beginning of long capillary column and ground to end with column itself negatively charged
- Then the free cations in solution are pulled towards anode at end and this generates bulk flow of liquid = EOF
- pos charged pulled to anodes faster then those pulled by bulk flow, and neg charged biomolecules pulled to anode slower then bulk flow = separation of biomolecules
Why do you need a microfluidic mixer?
Due to the very small channel diameter, Reynolds number is very low, leading to strict laminar flow.
What are the benefits of paper-based lab-on-a-chip over other silicone- or polymer-based lab-on-a-chip?
- Lower in cost, much thinner, easier to fabricate, and easier to use
- Only difference between paper LOCs and typical LOCs is use of paper in lieu of silicon
substrate - Doesnt require electroosomotic flow neither external pumping because fluid flows through via capillary action
What is PCR? How is it conducted?
Polymerase chain reaction is the process of amplifying the amount of target DNA sequence from the sample, and the production of target dsDNA can easily be monitored by adding SYBR Green I dye to the PCR mixture. The target antigen is added to the resulting mixture, and only a very small portion of candidate nucleic acids are able to bind to the target antigens. The resulting nucleic acid-antigen complex can be filtered, precipitated, or captured with gel, to choose the candidate molecule that possesses the best binding performance. The amount of chosen molecule is again amplified by PCR. The whole cycle is repeated more than 10 times to select only the best ones
Describe lateral flow assay (LFA). It is essentially a simplified, automated version of ELISA. What are the differences?
Basically, it is a simple to use diagnostic device used to confirm the presence or absence of a target analyte. The most commonly known type of lateral flow rapid test strip is the pregnancy test. LFA have a lower sensitivity over ELISA and is incapable of producing quantitive readouts
Compare LAMP and PCR.
LAMP is ideal for rapid, field-based diagnostics without sophisticated equipment, while PCR is more suitable for laboratory environments where precise temperature control and detailed analysis are required.
Fluorescent dyes that also function as bioreceptor
- DAPI
- Ethidium bromide
- SYBR Green I
Briefly explain how electric current is generated (or altered under applied voltage) from the GDH-PQQ detection of glucose.
Glucose is oxidized to gluconolactone in the presence of GDH-PQQ, while GDH-PQQ itself is reduced. Reduced GDH-PQQ can lose its electron to the electrode to be oxidized back to its active form. This oxidation-reduction cycle generates and consumes electrons, generating or altering electric current.
2 reasons why GDH-PQQ is de facto industrial standard in glucose sensing
Oxygen is not required.
Faster electron transfer rate.
Washing steps in ELISA can be simplified by using a microchannel. Briefly explain how it works.
The continuous flow through a microchannel makes the rinsing very effective.
Briefly explain why you cannot position the spectrophotometer at 0°
At 0°, the light detector will pick up both excitation from a light source and fluorescent emission from urine. At 90°, however, only fluorescent emission from urine will be picked up.
Do you need it for a paper-based lab-on-a-chip?
No, as the liquid is flowing through paper fibers, inherently generating cross-flows.
