In an article published in the prestigious Accounts in Chemical Research, Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., and his sensor team led by Senior Staff Scientist Pawan Jolly, Ph.D. in the Wyss Institute’s Bioinspired Therapeutics & Diagnostics Platform, describe the development and evolution of their eRapid affinity-based electrochemical sensor platform that is beginning to be applied to enable versatile and multiplexed low-cost point-of-care (POC) diagnostics for different hard-to-detect diseases and disorders.
This video explains the basic principles of eRapid as an electrochemical sensing platform, and how it could be used for low-cost, multiplexed detection of a wide range of biomolecules for diagnostic applications. Credit: Wyss Institute at Harvard University
Answering the call for multiplexed POC diagnostics
The accurate diagnosis of many diseases, such as sepsis, myocardial infarction (MI), and traumatic brain injury (TBI), depends on results from a series of diagnostic assays that measure the levels of multiple biomarkers. While all of those biomarkers are associated with the disease, obtaining a result for only one of them is often not a strong enough indicator of the disease – only a combination of multiple tests can create the confidence to make an unambiguous diagnosis.
This problem is further magnified when an early and timely diagnosis is needed in POC settings remote from clinical laboratories – where diagnostic tests are usually performed and analyzed. Ideally, health care workers and even non-professionals would have simple, robust, and multiplexed POC diagnostic tests at their disposal that could give them rapid results for several biomarkers and thus, the opportunity to make meaningful decisions and to respond more quickly with the appropriate measures. However, “such diagnostic capabilities do not exist yet in clinical or at-home settings and real-world POC tests would not only need to be ultra-reliable but also easy-to-manufacture at low costs,” explained first-author Sanjay Timilsina, Ph.D., who works as a Postdoctoral Fellow on the Wyss’ eRapid team.
In principle, electrochemical sensors engineered to detect specific biomolecules could make multiplexed POC diagnostics a reality. Electrochemical sensors are already used to detect toxic gases, oxygen and other air-borne molecules, for example, breathalyzers, respiratory carbon dioxide sensors, and carbon monoxide sensors. Once a gas reaches one of the sensor’s electrodes, called its working electrode, a chemical oxidation or reduction reaction happens depending on the gas to be analyzed. For instance, oxygen is reduced to water, which causes the flow of electrons to move from the counter (or reference) electrode to the working electrode, and thus creates an electric current that can be measured and is proportional to the concentration of oxygen in the gas.
However, only a few electrochemical sensing methods have been developed for clinical biomarkers with at-home glucose meters for diabetic patients being the best example. Glucose is highly abundant in blood and can be detected with a simple enzyme reaction. It is also a small molecule, which allowed engineers to cover electrodes with semi-permeable membranes that are permeable to glucose but prevent larger molecules from reaching the electrodes. However, most clinically relevant biomarkers, including proteins, are larger in size and cannot be measured using this approach.
In addition, they fall into the size range of many “biofouling agents,” a conglomerate of cells, proteins, and other biomolecules contained in blood, plasma, urine, and saliva that binds to sensor surfaces and renders them useless. Biofouling hinders the flow of electrons, causes background currents or electronic noise unrelated to the detection of the target biomarker, and can prevent the sensor’s biomarker detection agent from binding its target. Biofouling thus has been a major challenge to the development of electrochemical diagnostic sensors.
A second major challenge to developing multiplexed electrochemical sensor systems with high affinity to multiple biomarkers is posed by the relative dearth of affinity agents like biomarker-specific bio-receptors, such as antibodies, which need to be used in pairs to enable the specific detection of individual biomarkers. In addition, for the parallel detection of multiple biomarkers with arrays composed of multiple sensor elements, bio-receptor pairs must not produce cross-signals that interfere with electrodes of neighboring elements.
Solving the biofouling problem
The foundation of the eRapid platform was the team’s development of an antifouling nanotechnology-based strategy that could protect electrochemical sensors from the various fouling agents contained in blood and other body fluids. In a Nature Nanotechnology article, the team reported a simple coating that created a three-dimensional porous matrix on the sensor surface consisting of a network of conductive nanomaterials composed of gold nanowires or gold nanoparticles that were crosslinked by the chemical glutaraldehyde to the large biomolecule, bovine serum albumin (BSA).
These conductive matrices enhanced the transfer of ions through their nanopores to the electrode surface, while repelling biofouling agents. Some of the repulsion is due to the small pore sizes of the matrix, while the crosslinked BSA molecules repel albumin proteins that are present at an extremely high concentration in blood. As proof-of-principle, the team demonstrated that a version of their biosensor engineered to bind the inflammatory cytokine interleukin-6, could sensitively detect this target biomarker in unprocessed blood serum and, importantly, that almost 90% of the electrochemical signal was preserved even one month after the assay was performed. This is in contrast to other coatings being explored to prevent biofouling, which lost their protective effects in a few hours.
“Having created this antifouling strategy was game-changing for us in that it demonstrated that it is possible to create effective antifouling surfaces for diagnostic electrochemical sensors, and it marked the beginning of a multi-disciplinary journey crossing through very different areas of engineering, materials science, molecular biology, and medicine,” said Ingber. “It immediately breathed life into our much grander vision of an entirely new diagnostic platform with electrochemical sensing capabilities for various disease applications,” said Ingber who leads the Bioinspired Therapeutics & Diagnostics platform, and also is the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS).
From anti-fouling coating to versatile sensor platform
Following up on their initial success, the team further optimized the composition of the coating. Metal nanoparticles, such as the gold nanoparticles in their original coatings can oxidize molecules contained in blood and other biological fluids, which can create unwanted electrochemical background signals, or non-specific noise, how engineers call it. The high costs of gold nanoparticles and gold nanowires also stood in the way of the researchers’ goal to be able to manufacture diagnostic electrochemical sensors at low cost.
In their second study, which was published in Advanced Functional Materials, they overcame these issues by replacing gold with graphene oxide as a conductive material in their coating. Indeed, the graphene nanoflakes improved the coating’s anti-fouling properties even further. Exposing this next-generation electrochemical sensor to blood serum, plasma, or whole blood for one hour didn’t affected its electrical properties; and even constant exposure to human blood plasma over nine weeks only minimally reduced the electrical current they could carry. “The robustness and stability of our sensors also meant that the diagnostic binding assays can be performed at the patient site, and then the chips can be transferred to a centralized lab where the electrochemical signals can be measured much later, which could be important in under-resourced settings,” said Timilsina.
At the time, the team took their basic eRapid technology for a first real-world test-drive. In light of the large gap in diagnostic capabilities in the COVID-19 pandemic, they saw a unique opportunity and started to collaborate with a commercial partner in Australia, using eRapid technology to design a specific COVID-19 diagnostic assay, which has advanced to first-in-human trials with patient samples conducted at Harvard University and a license of the technology under the COVID-19 Technology Access Framework.
In parallel, after having optimized their nanocomposite coating, the team in a collaboration with the University of Bath that was spearheaded by first-author Uroš Zupančič, Ph.D., who was visiting Ingber’s group, developed their first multiplexing approach focusing on the diagnosis of sepsis. This disease cannot be rapidly and accurately diagnosed, and puts at least 1.7 million adults who develop it every year in America alone at risk of losing their lives.
The researchers leveraged the multiplexing capabilities to their eRapid platform to develop parallel acting sensors with working electrodes for the three sepsis biomarkers procalcitonin (PCT), C-reactive protein, and pathogen-associated patterns (PAMPs), whose levels variably rise in patients with sepsis, and currently are determined with the help of specific ELISA assays. In principle, a bio-receptor molecule that is specific for one of the biomarkers, such as an antibody or, in the case of PAMPs, the Wyss Institute’s engineered versatile FcMBL pathogen-capture protein – which can bind more than 120 different pathogen species and toxins, including the most prevalent sepsis-causing pathogens – is attached to the coating of an individual sensor element of a sensor array for multiple biomarkers. After the bio-receptor has bound its target biomarker molecule, a second bio-receptor that binds to a different part of the same biomarker molecule and which is linked to an enzyme is added to the complex (using two bio-receptors for one biomarker significantly increases the specificity of assay and allows the binding event to be amplified). A precipitate formed by the enzyme from a chemical substrate is deposited locally on the coating and increases the current of ions to the working electrode, thus enabling the sensor to register a chemical binding event as an electronic signal.
The researchers demonstrated that the multiplexed electrochemical sensor produced responses within the clinically relevant range without interference between the different insulated sensor elements. Further clinical development of eRapid for simultaneous biomarker measurements could therefore deliver a much faster and more accurate sepsis diagnostic assay that could be used at the POC or in physician’s offices with the potential to save many lives in the future. Toward this goal and other fast-acting diagnostic applications, the team coupled one version of a sepsis-specific sensor with a microfluidic system that allows the diagnostic process to be automated, thus making the handling of future POC devices much more user-friendly, and consistently producing results from very small blood samples in a much shorter time. This step enabled them to shorten the turnaround time for providing accurate measurements of a sepsis biomarker from almost an hour to a mere seven minutes, which is key for approaching future markets with a user-friendly diagnostic concept.
“This speed was unprecedented in the field and scientific literature, and the progress we had made thus far opened the technology up to designing POC diagnostics with typically required testing times of under one hour and longer-term applications in which biomarkers have to be continuously monitored,” said Jolly. “At that point, we were confident that we had a versatile and robust platform in hand that can be functionalized to detect a large range of analytes from small molecules, such as histamine and cortisol, to large proteins and antibodies.”
In more recent electrochemical sensors generated in the eRapid platform, Ingber’s team also created the opportunity to detect myocardial infarction and brain concussion by determining the levels of multiple biomarkers. Moreover, in additional unpublished work performed in collaboration with the groups of Wyss Core Faculty members James Collins, Ph.D., and David Walt, Ph.D., the eRapid team engineered electrochemical sensors for COVID-19-specific POC applications that simultaneously detect SARS-CoV2 RNA using CRISPR technology, and antibodies produced by infected individuals.
Streamlining eRapid sensor production
An important attribute of a diagnostic platform is the ease, consistency, and speed with which testing devices can be manufactured, and how well their components can be stored. In a third study, which has been deposited as an article on the medRxiv preprint server and awaits publication in a scientific journal, the researchers engineered these features into the eRapid platform as another key step in its evolution.
The use of graphene nanoflakes in the team’s advanced anti-fouling coating, in principle, is better compatible with mass manufacturing and long-term storage of electrochemical devices. Use of graphene also provides a ~99% reduction in the costs for the material when compared with first-generation gold nanowire and gold particle-based coatings. Initially, it took about 24 hours for the coating to settle on eRapid sensors; however, with a much-simplified ‘dip coating method’ developed by the team, they are now able to complete the coating process in less than a minute. “The approach also can be used with preassembled coating mixtures after they have been stored for at least five months at room temperature, which maintain more than 85% of the original electrochemical response levels,” said Timilsina who also is the first-author of the pending dip coat-engineering study.
Fabricating eRapid sensors using the new dip coating method allowed the bioengineers to produce their sensors for the electrochemical detection of multiple biomarkers of myocardial infarction and brain concussion with unprecedented sensitivity and selectivity. “This new rapid and straightforward method really opens the door for commercial mass-manufacturing of electrochemical sensors with high efficiency antifouling coatings for multiplexed POC diagnostic applications,” said Jolly.
From solving the biofouling problem for electrochemical diagnostic sensors onwards, the eRapid electrochemical sensor technology has been advanced step-by-crucial step by the team approaching their vision of a mature diagnostic platform that is ready to tackle numerous real-world medical challenges.