The file mentions that SPR is the workhorse in many labs today for studying protein binding.
That is very true.
SPR is widely used to measure:
From these:
K_D = rac{k_{ ext{off}}}{k_{ ext{on}}}
This is one of the biggest strengths of SPR compared with older equilibrium-only methods.
The basic principle is:
The lecture text says the gold plate can have different coatings, such as Protein A to capture antibodies.
That is correct.
For example:
So yes, the surface is often specially prepared depending on the experiment.
This is the deeper theory:
Light is shined at the gold film under a certain angle.
At one specific angle, the light excites surface plasmons.
A surface plasmon = collective oscillation of electrons on the gold surface.
Because of this, reflected light intensity drops at that angle.
When molecules bind to the surface:
That shift is proportional to how much mass binds.
So SPR measures change in refractive index near the gold surface.
Now to the main topic you asked about.
This part is very important.
The file says BLI gives the same kind of results as SPR, but the principle is different.
That is exactly right.
Both techniques measure binding kinetics and affinity.
But the physical principle is different.
Your understanding is already very close.
Let’s refine it.
BLI uses a biosensor tip / small chip.
The tip has:
The protein is immobilized on this sensor surface.
Then the sensor tip is dipped into wells containing ligand.
This is why BLI instruments often look like plate readers.
This is the key part.
Light is sent down through the sensor.
It reflects from two surfaces:
These two reflected light waves interfere with each other.
This is called interference.
If the biolayer thickness changes, the path length changes.
That changes the interference pattern.
This appears as a wavelength shift.
That is exactly what the file means by:
change or shift in the wavelength of reflected light
This is the part many students find confusing.
You asked:
when it touches the biolayer, it changes wavelength of reflected light — what does it mean?
Excellent question.
Strictly speaking, the wavelength of the light source is not physically “changed” in the usual sense.
What changes is the interference spectrum peak position.
Think of it like this:
The reflected light forms peaks and valleys.
When molecules bind, the biolayer gets thicker.
That shifts the interference pattern.
So the instrument reports this as:
Delta lambda
(change in wavelength position)
This shift is proportional to bound mass.
So:
A good mental picture:
Imagine two light reflections bouncing from two mirrors.
If one mirror moves slightly, the reflected waves no longer align the same way.
The interference pattern shifts.
That is essentially what BLI measures.
The “mirror movement” here is caused by molecules binding to the sensor.
Yes — absolutely.
This is an important correction.
They measure similar things, but via different physics.
Measures:
Signal comes from:
Measures:
Signal comes from:
Here is the simplest comparison:
| Technique | Principle | Signal source |
|---|---|---|
| SPR | Surface plasmons on gold | angle / refractive index shift |
| BLI | Optical interference | wavelength shift |
| Output | binding kinetics | binding kinetics |
So yes, different physical principle, but same biological interpretation.
Because both techniques track binding vs time.
That means both can produce:
For example:
During association:
ext{Protein} + ext{Ligand} ightarrow ext{Complex}
signal increases
During dissociation:
ext{Complex} ightarrow ext{Protein} + ext{Ligand}
signal decreases
This gives the sensorgram.
The file mentions their lab has BLI instead of SPR.
Common reasons:
So neither is “better” universally.
It depends on the experiment.
You wrote:
when it touches the biolayer
Slight refinement:
It is not the light physically touching and changing the layer.
Rather:
So the binding event causes the optical change.
Your intuition was good — it just needed slightly more precise wording.
The most important idea:
more binding = thicker layer = larger optical signal