A new micrometer-thick porous coating with unrivaled biomarker detection capabilities

Aging populations and the trend toward more sedentary lifestyles in many parts of the world are thought to significantly increase the number of people living with multiple chronic conditions. Additionally, climate change, as well as changing land use and travel patterns, continue to increase the risk of infectious diseases that can emerge and spread locally and globally.

Being able to rapidly diagnose the presence and progression of all of these diseases poses a growing challenge to healthcare systems, one that can only be addressed with effective point-of-care (POC) diagnostic tests. , beyond the doctor’s office and advanced medical care. facilities.

POC testing has brought many benefits to people during the COVID-19 pandemic, but this approach needs to become applicable much more widely and allow doctors and patients to delve deeper into pathological conditions. Current POC diagnostic technologies measure only a single disease biomarker or sometimes multiple biomarkers belonging to the same class of molecules, such as different RNAs, proteins or antibodies.

However, measuring multiple biomarkers from different molecular classes could provide more comprehensive information about the state a disease is in, its severity and progression over time, and even account for differences from person to person. different in the way it develops.

Electrochemical biosensors, which convert a chemical signal in the form of a biomarker present in a small sample of biofluid, such as blood, saliva or urine, into an electrical signal whose intensity corresponds to the detected quantity of the biomarker, could provide the answer to many POC diagnostic problems.

In principle, several sensors for different biomarker molecules can be combined in multiplexed sensor arrays and, more importantly, combating “biofouling”, the formerly inevitable destruction of electrode surfaces by non-specific biological molecules contained in the samples, has become avoidable thanks to the engineering of thin antifouling coatings were developed at the Wyss Institute at Harvard University.

Today, the Wyss Institute research team, in collaboration with several collaborating institutes in Korea, has taken electrochemical diagnostic sensing a critical step toward broader application by developing a novel porous nanocomposite antifouling coating. ‘a thickness of one micrometer, the diameter of a bacteria, approximately 100 times thicker than previous coatings.

The increased thickness of the coating and its porous network have enabled the incorporation of a much higher number of biomarker detection probes into the sensors, and thus, sensitivities up to 17 times higher than previous sensors, best in class, while also offering superior antifouling capabilities. .

In their proof-of-concept study, researchers built sensors that combine the ability to detect COVID-19-specific host nucleic acid, antigen, and antibody biomarker targets in clinical samples with sensitivity and high specificity. Their findings are published in Natural communications.

“Our new thick porous emulsion coating directly addresses critical barriers that currently prevent the widespread use of electrochemical sensors as core components of comprehensive POC diagnostics for many conditions,” said final author and Wyss founding director Donald Ingber, MD, PhD.

“However, beyond this, it could also open new opportunities to develop safer and more functional implantable devices and other healthcare monitoring systems across multiple disease fronts. Overcoming biofouling issues and sensitivity are challenges that impact many of these efforts.”

Ingber is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital and the Hansjörg Wyss Professor of Bioinspired Engineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.

Thicker coating, better detection

In 2019, the Wyss Institute Electrochemical Sensor Project published its landmark first paper reporting the first antifouling coating with unprecedented biosensing capabilities.

In a series of critical follow-up studies, the team increased the potential of electrochemical sensing by advancing the nanochemistry of coatings to make electrodes even more sensitive to biomarkers, adding significant multiplexing capabilities, and developing cost-reducing manufacturing methods.

The most advanced biosensors the team designed on Wyss’ eRapid platform had a set of features that already enable their translation into certain clinical settings.

However, the coating method used by the team exposed the entire sensor die to the nanocomposite solution and only allowed a relatively thin coating of about 10 nanometers to form over the entire surface of the sensor, which limited the functionality of the sensors in several ways.

For example, the thin diameter of the coating limited the maximum amount of probe that could be loaded onto it, which becomes particularly critical in larger multiplexed sensors that still need to operate with small sample volumes and even more so in efforts aimed at to miniaturize multiplexed sensors for their use. in portable POC diagnostic devices.

“In this new study, we found an entirely new solution to this problem, which resulted in a coating that is 100 times thicker. Our new approach leverages an inkjet printing method that allows us to apply this coating thick very locally on an individual sensor.” said Pawan Jolly, Ph.D., former principal scientist at Wyss, who was instrumental in the evolution of the eRapid platform.

“This opens up new possibilities: firstly, we can include much higher quantities of biomarker detection probes in the coating and, in the future, sensors in complex arrays can be addressed individually by applying nanocomposite chemicals specifically to them. adapted to specific applications.biomarker modalities.

Instead of literally dipping the electrochemical electrodes in a coating solution, as they did for their previous generation of sensors, the researchers printed a layer of a dense oil-in-water emulsion through a fine nozzle onto the electrodes. After the tiny oil bubbles evaporated, a 1 micrometer thick coating remained on the electrode surface, consisting of cross-linked polymer molecules from the blood protein albumin and containing interconnected pores and gold nanowires conductors of electrons.

“The porous network of this nanocomposite coating significantly increases the surface area that can be used to attach specially designed biomarker detection probes, and which is at the same time accessible to fluid samples. As a result, the detection sensitivity is significantly increased. ” explained first author Jeong-Chan Lee, Ph.D., a postdoctoral researcher on Ingber’s team.

“Additionally, nozzle printing allows us to pattern the emulsion exclusively on the biomarker sensing working electrode while keeping the neighboring reference electrode contained within each free sensor, reducing non-specific electrical noise and improves the specificity of our measurements.”

From COVID-19

The team reused a previously developed combination of detection reagents for three COVID-19-related biomarkers to model a sensor electrode array using their newly developed coating technology: a CRISPR-enabled sensor for an RNA of SARS-CoV-2, a specific sensor for a SARS-CoV-2 capsid antigen and a sensor for a host antibody against the virus.

Tested with a collection of patient samples, the new sensor produced detection sensitivities improved by 3.75 to 17 times compared to a previous one made with the same detection systems and the better non-porous and much thinner coating of the team. It also distinguished positive samples from negative samples with 100% accuracy (specificity).

“Electrochemical sensors with this next-generation coating would be ideal for monitoring viral outbreaks, vaccine responses and understanding correlations between various biomarkers during viral infections and, in the future, could also be used for other diseases.” Lee said.