How a biochemist copied nature to engineer DNA towards heart failure detection

Problematic

Some diseases require treatments that can have adverse effects on other organs. This is particularly the case of heart failure, which can be treated with a class of drugs, angiotensin-converting enzymes (ACE) inhibitors, such as Ramipril or Quinapril. Unfortunately, these drugs can potentially be toxic to the kidneys. In treating patients with heart failure, doctors obviously do not want to cause them new health problems. Thus, to circumvent this problem, they will prescribe such drugs, but using suboptimal doses. This allows them to control and alleviate the initial problem of heart failure without causing them complications to the kidneys. However, such a solution is ineffective because, in addition to limiting its action on heart failure, this therapeutic limitation requires patients to complete frequent blood tests to monitor the effect of the medication and verify that the other organs are not affected.

At the same time, since 2011, people over 65 have been more numerous than people aged 0 to 14 in Quebec. This demographic disruption comes with its own set of challenges: the increase in the average age of society has serious consequences for the health system. This demographic change is of particular interest in the case of heart failure since the prevalence of contracting this disease after 55 years is 30%. Considering the increased congestion experienced by hospitals in Quebec since 2012, an alarming problem caused by an increasing influx of patients suffering from heart failure and requiring blood tests should be expected.

A promising solution to this problem would be to switch the collection site from hospitals to homes and clinics. Over the past decade, significant efforts have led to the development of technologies that make diagnostic tests simpler to operate and at a lower cost: DNA biosensors.

I sat down with Dr. Alexis Vallée-Bélisle, a biochemist turned engineer who is developing a biosensor capable of detecting hematological biomarkers relative to heart failure and kidney failure.


Overview

Dr. Alexis Vallée-Bélisle is the principal investigator of the Laboratory of Biosensors & Nanomachines and the Canada Research Chair in Bioengineering and Bio-nanotechnology. He completed his bachelor in biochemistry and continued in a very theoretical branch during his master and Ph.D., studying the folding mechanism of proteins. It abled him to gain a unique perspective on a variety of complexes biochemistry mechanisms. He viewed the biochemistry as natural nanotechnologies. Following that idea path, he completed his post-doctorate at Santa-Barbara in bio-engineering and more precisely on electrochemical DNA biosensors.  During this fellow, he was able to link strategies used by nature to the detection domain. The goal of his laboratory is inspired by the biochemical background and from efficient nature mechanism: develop simple diagnostics methods.

Dr. Alexis Vallée-Bélisle Director of the Laboratory of Biosensors & Nanomachines Canada Research Chair in Bioengineering and Bio-nanotechnology

What is the principle behind the DNA-based electrochemical sensor and why is DNA such a promising molecule for sensor applications?

 Biosensors are based on the philosophy behind glucose sensors, which is to use a small amount of blood through which the concentration of a chosen target can be detected. In the glucose sensor, the glucose level is monitored in real time. This sensor revolutionized diabetes treatment. As of now, sensors are only used to track this disease, but they have huge potential.
To be functional, the biosensor must have three distinct components: (i) a specific recognition element, (ii) a sensitive signal element and (iii) a conductive surface.

Conductive surface

The first component of the sensor is the gold electrode. This metal is of choice because it has a limited reaction window, it will only react with specific molecules and it allows a conductance of electricity. We can, therefore, apply and measure an electrical current on the gold surface. At the same time, it serves as a support for the DNA molecules.

Signaling element 

Signaling in the biosensor is done by electrochemistry. Electrochemistry represents the relationship between electricity and measurable and quantifiable changes at the chemical level. In fact, this branch of chemistry measures and observes the transfer of electrons from one molecule to another in the presence of an electrical circuit. Some electroactive molecules named redox labels have a better ability to transfer electrons because of their three-dimensional conformation. Their electron transfer is quantifiable.

 Specific recognition element 

DNA is viewed as only genetic material, but it is much more than that. Its simple yet highly specific hybridization mechanism between nucleobases is the basis of these biosensors. A significant breakthrough in the field of DNA-based biosensors is the development of a method for the design of DNA sequences capable of recognizing virtually all constituents of the living, be it a small hormonal molecule or a huge viral molecule. To do this, scientists have developed a method of artificial creation of DNA mimicking natural selection.

These DNA molecules are called “aptamers”.

What is fascinating is the movement these DNA molecules have. Drawing inspiration from enzymes in nature, aptamers are engineered to have a switch mechanism with an “ON” structure and an “OFF” structure. In this vein, DNA biosensor relies on DNA more as a material to create a switch mechanism that will be mobile. DNA is not used as a recognition element but as a mechanism to create nanomachines.

How does your prototype sensor work?

We are developing a sensor specifically applicable to the case of heart failure and kidney failure. 3 blood markers are indicative of both these conditions: potassium, creatine, and urea. These are the 3 markers that are targeted by this sensor. This sensor would, therefore, have 3 separate electrodes that would receive blood and it would display the blood concentrations for each target analyte.

The sensor foundation is the pre-processed gold electrode that is connected to an electrical circuit that sends an electrical impulse and measures the exchanges on the gold surface. An aptamer will have been synthesized beforehand to recognize the targeted analyte, and this aptamer will be separated into its 2 DNA strands. The recognition DNA chain will then be bound on this gold surface; afterward, this DNA strand is hybridized by its complementary strand. The particularity of this complementary strand is that it comprises a redox label linked to one of its ends. For this sensor, the molecule used is methylene blue. As a result of hybridization, methylene blue will begin to transfer electrons with the gold surface. Once the two chains of DNA are linked, the aptamer will be able to recognize its target. Our electrode will be ready to measure our biomarker. Thus, when the patient’s blood is in contact with the gold electrode, only the targeted biomarker will bind to the aptamer. This will create a structural change and change the 3D conformation of the DNA. This shift will cause methylene blue to shift. The latter will, therefore, be further away from the gold surface and there will be a reduction in electron transfer. The proposed biosensor will monitor and study this transfer variation, and this variation will then become quantifiable and allow the amount of analyte in the blood to be calculated.

What were the biggest challenges associated with biosensors?

Even before stability issues or any problems associated with the commercialization, there is one major issue common in all biosensors. It is difficult to create a biosensor with a robust design that will perform accurately in whole blood. It is very challenging to build a system that will produce a specific signal related to the target and not to the background of proteins contained in the whole blood. Biosensors that use a mass variation or charge variation on the gold surface as an indicator have this obstacle. Proteins enclosed in the whole blood will deposit on the gold surface. The signal generated will then not be a good indicator of the target, as it will have been contaminated with the other blood constituents. The use of DNA and aptamers is a novel solution to bypass the non-specific binding.

What other applications could this sensor have?

A biosensor that uses DNA and aptamers are the foundation of a diagnostic tool that has immense potential. The same principle can be used to detect other targets. There is a variation of this principle that enables the sensor to detect a larger variety of molecules. If the target does not have a specific aptamer that is able to bind to it, there can still be an approach to detect and quantify them.

Smaller markers can be detected by steric hindrance using antibodies in a competitive assay. If the target is present in the blood, they will bind to the antibody and the small signaling DNA will be able to reach the electrode surface and hybridize efficiently to a majority of the complementary recognition DNA strand. This will generate a large electrochemical current because the redox labels will be closer to the electrode’s surface. If they are no target analyte in the blood sample, the signaling stand will remain bound to the antibody, thus reducing significantly the ability of this large DNA complex to hybridize on the electrode surface due to steric hindrance. This will conclusively lead to a low electrochemical signal.

In other cases, it possible to use the pH. This is the case for urea. In the presence of the enzyme urease, urea will create ammoniac and thus rendering the solution more basic. This modification in the pH will reduce methylene blue’s activity and consequently the electron transfer. It is possible to quantify the concentration of urea by monitoring this change in electron transfer.

 

Fig. 1 Schematic representation of a competitive electrochemical steric hindrance hybridization immunoassay to detect small molecules.  In the absence of a specific analyte in the sample (bottom), the signaling strands remain bound to the antibody, which significantly reduces the ability of this large DNA complex to hybridize on the electrode surface due to steric hindrance. This ultimately generates a low electrochemical signal (green curve). In the presence of target analytes, the latter bind to the antibody and the small signaling DNA is able to reach the electrode surface and hybridize efficiently to most complementary capturing strands, thus generating a large electrochemical current (black curve) by bringing redox labels near the electrode’s surface.

 

Can this technology be made commercially for widespread use?

This research is currently being developed on a small scale in the laboratory. Once this sensor is completed et perfected, all the electrode preparation steps will be done in advance. A patient with heart failure will then be able to simply use this technology by putting a small amount of blood in the intended area, and with the help of algorithms and computer programs, the electrical impulse will be transformed into a blood concentration that will be displayed for the patient. Patients with heart failure will be able to stay in the comfort of their homes to take blood samples. The accessibility of these blood tests will allow health workers to prescribe the optimal doses of the drugs against this pathology because they will be able to follow in real time the toxic effects on the other organs. For the general population, this will mean less hospital congestion, better access to care, and reduced health costs.


Reference:

Mahshid, S. S., Ricci, F., Kelley, S. O., & Vallée-Bélisle, A. (2017). Electrochemical DNA-Based Immunoassay That Employs Steric Hindrance To Detect Small Molecules Directly in Whole Blood. ACS Sensors, 2(6), 718-723.

 

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