0:33
My name is Karen Leirs and today I would like to show you how fibre optic surface plasmon resonance technology can assist in improved production and characterisation of biopharmaceuticals.
0:46
But before I dive into the technology and its applications, I would like to give a short introduction to the Biosensors group of KLU and where all this research is being performed.
0:59
So the Biosensors group started in 2005 and is led by Professor Jeroen Lammertyn.
1:05
We're quite a big group with research staff, postdocs, PhD students and lab assistants.
1:14
Over the years, we have obtained more than €37 million funding from both international and national funding agencies.
1:27
We have more than 280 scientific publications.
1:31
Our research is related, is both fundamental and applied and related to life science research tools and diagnostic devices.
1:42
We are a group that's also focused quite a lot on commercialization.
1:46
So we have a dedicated innovation manager, 2 patents portfolio, 1 spin off company and we have some multimillion bilateral contracts with industry.
2:01
Our network is extensive, more than sixty partners, academic ones worldwide and more than 50 companies.
2:14
So the mission of the Biosensors Group is to develop innovative in vitro diagnostic solutions and life science research tools that have a high application potential both in industrial and clinical settings.
2:31
So this ranges from biomarker discovery and bioassay development towards prototype validation.
2:42
So based on this mission, we have established actually 6 research lines.
2:47
So the first one is bioassay development and implementation, where we, for example, look into bioreceptor and surface chemistry selection, but also strategies for single for signal amplification.
3:03
Next, we have expertise in microfabrication, for example, surface modification of different types of surfaces and also clean room based fabrication.
3:15
Next, we are also developing tools, microfluidic tools for point of care tests to enable the implementation of complex bioassays at the point of care, but also micro sampling of body fluids and drug and vaccine delivery.
3:32
The last couple of years we're also working on DNA nanotechnology for improved biosensing by developing DNA nano tailored surfaces, ultra-sensitive DNA nanosensors and CRISPR enabled genome imaging.
3:52
Besides, we are developing microfluidic tools for single cell analysis to allow high throughput screening of single cells and spatial temporal analysis.
4:04
Finally, we're also working on optical biosensing like the fibre optic SPR technology that I will explain in more detail in a couple of slides.
4:16
These research lines are supported by some market pool emerging fields such as compound discovery, innovation in the agro-foods sector, the evident needs for pandemic preparedness, the continuous quest for wearable devices, for example, for disease management.
4:37
Also there’s a lot of research going on in the single cell multi-omics field and continuous improvements or continuous developments for improved bioreactor monitoring.
4:52
So now let's dive into the technology.
4:55
So fibre optic surface plasmon resonance, it's actually similar as the more well-known prism based surface plasmon resonance platforms like Biocor for example.
5:09
But here we work with an optical fibre that is coated with a gold layer and through which light is shined.
5:19
Now on the surface of this fibre there will be surface plasmons created similarly as the other SPR systems.
5:29
And this causes a dip in the reflectance spectrum at this resonance wavelength.
5:35
Based on interactions that are happening on the surface, this dip in reflective spectrum will shift and this is how we can monitor these binding reactions.
5:47
So the technology allows real time and fast detection of targets and it's very flexible because different types of targets can be actually detected.
5:57
It enables kinetic analysis and multiplexing.
6:00
It's sensitive and allows the detection directly in crude sample matrices.
6:08
This technology has been developed by our group what is at this moment commercialised by FOx Biosystems.
6:15
So they have a booth here as well.
6:16
So if you haven't done so, don't forget to go and check them out either today or tomorrow.
6:25
So as I mentioned, we worked on this technology already for several years.
6:30
So we have developed a lot of different applications and I want to show you some of these today.
6:38
Some of these applications will be directly implementable on the FOx device, FOx Biosystems and this you will see on the picture of the device that will appear on the slides.
6:50
Others are further developments that we did on our own in-house system.
6:56
So let's dive into the applications and let's start with the immune-based detection.
7:02
So first one is the therapeutic drug monitoring of biologics.
7:08
So multiple autoimmune diseases are being treated with biologics, but they're very important is that one dose does not fit all.
7:18
So it's very crucial that the reaction of the patient to the treatment is monitored and this is what we established here.
7:26
So we started from a COOH surface where we immobilised antibodies that are then specifically binding in this case on the TNF antibody in the patient sample.
7:40
We have actually shown that our test has the same performance as a lab based test and can be performed in different biological matrices such as serum, plasma, dried blood spots and even whole bloods.
7:59
In the next application we have shown that we can also do small molecule quantification by implementing a competitive assay.
8:08
So what we have done here?
8:10
We started again from the same COOH surface and now immobilised BSA molecules labelled with Progesterone.
8:18
These progesterone molecules, they compete with progesterone in the sample for binding to the antibodies that we add.
8:28
So the more progesterone there is present in the sample, the lower signal generation we will have.
8:36
Here we have shown that this works in both buffer and milk and benchmarked this against a commercial ELISA test showing again good performance.
8:51
Next application we actually changed to a different surface chemistry, namely NTA.
8:59
As you might have seen in the previous slides, with COOH surface we actually have a random distribution of our bioreceptor on the surface, meaning that some of these bioreceptors are actually not available for binding their targets because they are for example oriented in a wrong way.
9:18
This is not the case with NTA where we have actually an oriented deposition through the surface of our bioreceptor and this allows us to actually obtain multiplex detection on one single fibre probe.
9:36
As you can see here, we have two different bioreceptors on the surface that we then bring in contact with our sample so the target can bind and then we do a sequential signal discrimination and amplification of the bound antibodies.
9:55
In this case, we have also shown that the same surface can be used in serum and whole bloods and enabled as such kinetic profiling of antibody mixtures also with NTA surface chemistry regeneration is possible which can be of interest for certain applications.
10:18
So these were some of the examples of our immune-based detection that we have implemented on the FOSPR technology.
10:27
But we are also doing DNA-based detection.
10:31
And here in the first example, I want to start with another strategy to do actually oriented deposition or binding of the bioreceptors by in this case using DNA origami.
10:47
So here we have developed DNA origami structures. At certain positions strands protruding from the surface that can either bind the gold surface, the red ones here, or are actually bioreceptors for target binding.
11:06
And as such, we obtain fully controlled surface functionalisation and have also improved bioreceptor accessibility because we can fully tune the distance between our bioreceptors to perfectly match the targets and as such reduce, for example, steric hindrance.
11:28
We have shown different ways of making this origami structure.
11:33
So either the protruding strands on lateral surfaces or on the distal ends or even a completely different structure of DNA origami.
11:46
This showed that we can have sensitive detection and even no back filling needed to reduce our specific interactions that are otherwise occurring.
11:55
A completely different application is DNA detection using NAzymes.
12:04
So here we have developed an asymmetric PCR that when the target is present, actually generates DNAzyme-amplicons.
12:14
And it's these amplicons, well specifically this DNAzyme that can cut DNA strands that are on the fibre surface and as such release nanoparticles that then result of course in a signal change.
12:33
This similar concept can also be applied, for example using isothermal amplification techniques instead of PCR.
12:43
Last couple of years we are also working on the development of nanoswitches and their implementation on the fibre optic SPR technology.
12:54
So what we are doing here is we have a DNA strand on the surface that is actually an aptamer but at this point it's partially hybridised to another DNA piece that is bound to a nanoparticle.
13:08
And upon target binding, this hybridization will become loose as the target will bind its aptamer and as such we have a release of the nanoparticle.
13:19
This allows weight independent target binding and we as we have shown here that it can be used for different types of molecules like ATP, small molecule thrombin protein or DNA sequences and it's possible.
13:35
Well this concept is applicable in complex matrices such as plasma.
13:43
Now we even took this concept a step further and similarly as in the previous slides, we have here same concept with an aptamer and a hybridised region or hybridised DNA sequence to it.
14:00
But upon target binding now the particle is not released in solution, it's actually bound through this linker.
14:07
It stays bound to the fibre surface and what we have is a signal generation based on a spatial redistribution of this nanoparticles on the surface.
14:21
To actually obtain good signal here, we had to stain plasma coupling between these nanoparticles and the fibre optic sensor by reducing the gold layer.
14:35
So this is a thinner gold layer compared to the previous applications.
14:40
But with this application or with this strategy, we can actually do continuous sensing.
14:50
So as you can see here, we are detecting DNA targets in a continuous way.
14:57
So you can see we can follow up different concentrations of this DNA target in a continuous way.
15:02
And actually when we provide samples without any DNA present, we actually come back to the same baseline as we had before.
15:14
So showing that we actually have the same result without regeneration as we would have with regeneration.
15:22
Of course, here it's very important that this target binding is reversible.
15:27
But this is actually the good thing about working with DNA as a bioreceptor because you can fully tune it to fit actually what you need in the bioreceptor.
15:40
So as mentioned, we're working on different applications, but also we are further working on technological advancements of this technology.
15:53
And here we are for example, looking into sensitivity enhancement because this can be crucial for certain applications.
16:00
And we want to do this by creating localised electric fields by plasma etching the surface.
16:07
So as you can see here, we have a plasma etched surface.
16:10
So this is a rougher gold surface compared to the smooth surface that we have otherwise.
16:17
And you see we have an increased sensitivity which we also shown by three times increased binding shift upon target binding for example here shown for this thrombin assay where we have for the etched fibre indeed a higher signal shift.
16:36
Finally, we are also working on improved self-referencing of this FO-SPR sensors.
16:44
So here we want to do real time correction of the sensing signal for external and internal fluctuations.
16:53
So how we do we do that is we have our sensor probe where we have two zones, a sensing zone a and a referencing zone.
17:02
So it's on the same fibre, we have both zones.
17:04
And as such we can actually do this signal correction.
17:07
The aim here is to integrate this in a bioreactor via microfluidic sapling.
17:16
So I hope I managed to give you a flavour of all the different applications that we have done over the years with the FO-SPR technology.
17:27
But now maybe to come back to my title, how can FOSPR indeed improve biopharmaceuticals?
17:35
Well, we can follow up production status.
17:37
This can be for proteins, for DNA, for small molecules.
17:43
We can determine product quality safety by looking at cell viability, for example, via host cell proteins or the presence of microbial cells, for example, via small molecules.
17:58
We can perform kinetic profiling and we can enable continuous monitoring both in line and online.
18:07
So I want to thank my colleagues and a special thanks to Annelies, Claudia, Jalu, Pradana, Jihuan, Bernd, Jiadi and Devin because what I've presented here was definitely not all my own work.
18:18
So thanks to them for all this hard work and I'm happy to take any questions.
