It was an enormous pleasure and a privilege to chat with Peter – who we also realised is probably one of a NuNano’s longest standing customers, having first ordered from us in early 2017!

Peter’s story resonates with his delight and enchantment with the scanning probe mciroscopy and his commitment to pushing the boundaries of its known and accepted uses is truly inspirational.

When Peter Nirmalraj first said he wanted to use atomic force microscopy (AFM) to study proteins in blood for disease diagnosis five years ago, it was seen as a very ambitious experiment. It simply wasn’t the done thing. The thousands of red blood cells in a single drop of blood had researchers convinced the sample would be too noisy to produce data of any merit.

But Peter, by this time in his career a die-hard AFMer, was adamant it was worth pursuing as it was a label-free method capable of providing nanoscopic insights under standard laboratory conditions. For him, the current methodology of centrifuging blood into its parts, seemed limited.

“This approach struck me as the equivalent of only looking for your lost car keys in a dark car park under a streetlight because that's where the light is,” says Peter.

The centrifuge process, which removes the red blood cells (RBCs) to focus only on the easier-to-analyse component plasma, raised questions. Peter wanted to know whether the RBCs themselves could offer an alternative rich pool to screen pathological proteins.

AFM as a torch

“AFM felt more like a torch, enabling us to explore the whole parking lot. Moreover, red blood cells present a large area that could be ideal for AFM analysis – 8 microns across, concave and flat at nanoscopic length scales and 1.8 – 2 microns thick. And as RBCs are prevalently found in blood, there was a higher chance for the pathological and non-pathological proteins in the blood to stick to the lipid membrane-based surface of RBCs.”

To test this hypothesis, Peter set up a study in his laboratory at Empa together with clinicians at Kantonal Hospital St Gallen to investigate the surface of RBCs obtained from patients at various stages of decline in memory and cognitive health.

Nearly 100 patients were recruited as part of a single-blinded study, including control of healthy patients, and whole blood samples were taken from them at different time points. These samples were prepared as blood smears and sent to Peter's lab for AFM analysis. The first measurements were done on about 50 samples, and around 1,500 –2,000 red blood cells were measured per sample.

Disease stage correlations

Peter looked at the differences in morphology, quantifying the data before finally handing it over to the clinicians. What followed was an extremely nervous wait until the clinicians did the correlation.

When they came back to Peter they said, “Well this is fascinating. There is a very solid correlation between what you measured using AFM and it reflects the disease stages.”

“To me, that was the first step in identifying a physical biomarker for Alzheimer's disease and related pathologies,” says Peter, “with the differences in the size, shape, and overall prevalence of the protein aggregates on the surface of RBCs indeed reflecting the stages of decline in memory and cognition of patients.”

The role of the AFM was to provide new and complementary information on the disease stages, which the clinicians could use as one more piece of the puzzle before making the final diagnosis.

The research was published in the journal Science Advances.

Clinicians that work with Peter. From Left to Right: Dr. Ansgar Felbecker, Dr. Thomas Schneider and Peter Nirmalraj.

Clinical Relevance

But that wasn’t the end of the revelations from this study yet more completely new information came to the fore. An unexpected finding was that in patients over 75, the surface of the red blood cells had crystallographic deposits of proteins. Peter found this intriguing as, when magnified through the AFM, the fibrils were all aligned. However, when he showed the data to the clinicians, they were excited for a completely different reason: the data was telling them something else.

Elderly Alzheimer’s patients often suffer from asphyxia and looking at Peter’s data they realised a new explanation might be emerging.

“With the surface of the red blood cells having so many aggregates on it, the role of the red blood cells as a transport of oxygen becomes difficult and this could potentially influence the onset of asphyxia. Whilst this needs to be validated in a larger cohort of patients with similar age and cognitive health conditions, it is an exciting discovery,” says Peter.

This promising result convinced Peter that what we learn at the nanoscale has a certain clinical relevance. The clinicians began to appreciate the AFM results more, and now AFM is part of a long-term memory clinic study in Switzerland.

“We still combine AFM with traditional techniques like fluorescence-based methods to get a chemical signature, but the AFM data is fundamental,” says Peter.

“We aim to position the AFM as a powerful tool for early screening of slow but relentlessly progressing neurodegenerative diseases and not for rapid point-of-care type diagnostics.”

Study of a lifetime

The work has since expanded to studying cerebrospinal fluid where similar results have been found. Peter and the team are also working on a second phase, to devise studies aimed at investigating both blood and spinal fluid from patients who have received anti-amyloid drugs to see if protein aggregation landscape changes in body fluids. The study has also expanded beyond Switzerland, with Peter traveling to places like Brazil to visit clinics and collect samples from different demographics. He is keen to see if the findings hold across various ethnicities, which is critical for validating their work.

Peter recognizes that this line of study is the work of a lifetime, and he’s grateful to the management at Empa for supporting his continued focus on it.

From STM to AFM

Peter’s journey into working with AFM has been an organic one, sparked by an article he read at high school about scanning tunneling microscopy where the IBM logo had been made out of atoms. Fascinated he emailed the senior researcher who, to Peter’s surprise, replied in detail, encouraging him to pursue a career in surface science.

“Coming from Tamil Nadu, a state in southern India, where surface physics research wasn’t a common career path 20 years ago, it was a big leap.”

That leap took Peter to KTH, Sweden to do a master’s in nanomaterials and complete a research thesis based on ultra-high vacuum-based STM and then to Trinity College Dublin, Ireland, where he worked on conductance imaging AFM in the lab of Prof. John Boland to study charge transport in 1-dimensional and 2-dimensional material networks. After acquiring a Marie Curie postdoctoral fellowship, he moved to IBM research laboratories in Zurich, which is the birthplace of scanning tunneling microscopy. During his time at IBM, Peter realized the potential of scanning probe microscopy from studying energy levels of single molecules at the solid-liquid interface to larger and more complex molecules such as amyloid proteins

That drive took Peter to the Adolphe Merkle Institute, Fribourg where, using NuNano probes, he began to look at proteins implicated in the pathology of Alzheimer's.

“With this work, I made the shift to liquid-based AFM. We were observing the aggregation pathway of amyloid beta 40 and 42 proteins and were able to record and quantify the changes in their size and shape – which was interesting to observe as they are well-known shapeshifters.” More recently we have been able to extend this study to further understand the secondary nucleation pathway of amyloid-42 proteins at the solid-liquid interface directly on the surface of parent fibrils (https://www.science.org/doi/full/10.1126/sciadv.adp5059).

From there Peter moved to Empa, the Swiss Federal Laboratories for Material Science and Technologies, a laboratory dedicated to materials characterization research. He started his research group on Biosensing and functional surfaces in 2019 at The Transport at Nanoscale Interfaces Laboratory and began his most recent momentous journey into studying the role of protein aggregation and neurodegenerative disease progression.

Of working with scanning probe microscopy Peter says; “It’s addictive and I thoroughly enjoy working in the lab together with my small group of postdocs and PhD students, which allows me to stay hands-on with the research. I do not formally teach at a university because it would take me away from the lab, but I see the lab as my classroom where together with a highly motivated team we focus on understanding and solving one problem at a time.”