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Swiss Research Group Developing Electrochemical C-Reactive-Protein Assay for the Point of Care


NEW YORK (360Dx) – Researchers at the Swiss Federal Institute of Technology (SFIT) in Lausanne are using C-reactive protein as a biomarker to develop an assay that they believe could become part of a commercial test applied at the point of care to detect cardiac conditions.

The assay requires small sample quantities, and it could be inexpensively screen-printed from carbon, a cheap and plentiful material, Madasamy Thangamuthu, one of its developers and a researcher in the nanophotonics and metrology lab at Swiss Federal, said in an interview.

He noted that the test works well with serum and saliva, and when used with serum, it requires only a single drop of blood to operate. Further, he said, by employing a label-free approach — which avoids use of antibodies that are required for laboratory assays — the researchers were able to simplify the design and take even more cost out of building the assay.

In a study published recently in the journal Biosensors, the SFIT researchers noted that CRP is being investigated as a biomarker for several cardiac conditions, including myocardial infarction, stroke, atherosclerosis, peripheral arterial disease, and sudden cardiac death.

Their immunoassay consists of a single strip, similar to a pregnancy test used in the home, and has three connected electrodes combined in a compact form that can easily be slipped inside a pocket, Thangamuthu said.

His group expects to soon speak with in vitro diagnostic developers about potentially commercializing the test, but before that happens, they will need to perform clinical testing on a broad set of samples, he said.

A C-reactive protein commercial test used in the home would be a unique and useful application, Shekhar Bhansali, Alcatel Lucent professor and chair of the department of electrical and computer engineering at Florida International University, said in an interview. Bhansali, who is developing a test to detect cortisol at the point of care but is not connected with developing the SFIT C-reactive protein test, said that such tests could provide several important advantages over lab tests and would significantly improve patient care.

In routine clinical laboratories, serum CRP is usually estimated by turbidimetric, microplate reader, and enzyme-linked immunosorbent assays. The tests are well established, reproducible, and show sufficient sensitivity, but they are not suited to use at the point-of-care because they are time consuming to execute, relatively expensive, require trained personnel, and are only available in laboratories.

In measuring C-reactive protein, "a point-of-care approach allows you to get a much better understanding of the underlying disease profile and enables better disease management," Bhansali said.

If the researchers are eventually successful in commercializing a point-of-care test, patients would be able to conveniently create a map of C-reactive protein levels over time that's reflective of underlying conditions, he said. Patients and their physicians, through computer connectivity, would be able to monitor the results and act on them immediately, an advance over lab testing that provides a single data point after you wait, possibly for days, for results. 

Because of the potential benefits of point-of-care testing, which also include technical and cost advantages, researchers are looking to replace laboratory-based ELISAs and immunoblot assays that must integrate both primary and secondary antibodies to detect analytes of interest, Thangamuthu said.

Conventional sandwich-style laboratory ELISAs and immunoblot tests require both types of antibodies to capture an antigen or analyte of interest and generate a signal, sometimes a fluorescent one, that can be measured.

The Swiss Federal point-of-care prototype, by contrast, requires only a primary antibody. Within its tiny design, it consists of a sensor strip with a screen-printed carbon electrode modified with anti-CRP functionalized gold nanoparticles. At the heart of the device is its electrochemical sensor, Thangamuthu said, which measures a reduction in current that occurs when an immunoreaction between the C-reactive protein antigen in the sample and C-reactive protein antibody in the test blocks electrons.

Under optimal conditions, the immunoassay measures CRP in a linear range from 0.4 to 200 nM with a detection limit of 0.15 nM and sensitivity of 90.7 nA nM−1. That translates to a low detection limit, high sensitivity, large detection range, and excellent repeatability and stability, the researchers said in their paper.

CRP has become important for early-risk assessment of cardiac diseases and within initiatives to reduce the number of deaths related to cardiovascular conditions, the researchers said. The marker is similar to cortisol in that it can also be an indication of many underlying conditions, including non-cardiac conditions, such a fibromyalgia, Bhansali said. 

Because of the potential for using the CRP test at the point of care, researchers have been investigating alternatives to laboratory instruments. They are using various technologies, including surface plasmon resonance, piezoelectric microcantilevers, quartz crystal microbalance technology, microfluidics, and electrochemical methods, the researchers said.

They noted that electrochemical immunoassays are promising, in general, because of their levels of sensitivity, lower detection limit, fast response, cost, ease of handling, and miniaturization. However, although extensive research efforts are under way, the development of a simple and high-performance CRP immunoassay is still challenging for point-of-care applications, the researchers noted.

Point-of-use diagnostics is an "up and coming" field, Bhansali said. Few commercial diagnostic tests that enable biochemical analysis on a continuous basis at the point of care are available because "it's not easy to pull it off," he said. "If somebody can do it right, it's going to be a very big deal."

Reducing an electrochemical point-of-care device to a suitable form factor and making the device so usable that patients don't compromise performance in providing samples or otherwise operating the test is an important consideration, he said.

There are many variables that need to be considered in a point-of-care test, such as variability in how people draw blood. Also, people live in widely different weather conditions, involving swings in temperature and humidity. These and other factors need to be considered so that they don't impact the performance of a test, he added.

Capacitive electrochemical immunoassays, although they have potential as hand-held point-of-care devices, suffer from non-specificity and background signals that limit performance, the SFIT researchers noted. Nonetheless, their label-free electrochemical immunosensors are showing commercial promise, and they can be adapted for the measurement of different clinical biomarkers, Thangamuthu said.

He noted that in prior work, he developed an electrochemical biosensor that the Indian military licensed and that measures levels of nitric oxide metabolites at the point of care in soldiers working at high altitudes who suffer from oxidative stress brought on by deprivation of oxygen.

In 2014, Thangamuthu with colleagues published a study in Sensors and Actuators A: Physical that described the use of another point-of-care cardiac disease marker, cytochrome c. They measured mitochondrial cytochrome c released during cardiomyocytes apoptosis, a condition connected with myocardial infarction, myocardial ischemia, and heart failure.

The researchers evaluated the performance of a miniaturized screen-printed electrode biosensor coupled with an instrument that consisted of a portable microcontroller and noted that the results compared well with those of a commercial electrochemical analyzer and standard ELISA.

Among other researchers developing point-of-care tests for cardiac conditions, University of Turku researchers have developed a proof-of-concept troponin I platform that could be further developed as a diagnostic system. The platform uses photoluminescent up-converting nanoparticle technology to achieve limits of detection that are comparable to that of the most sensitive troponin I lab-based assays, according to its developers.

Other researchers are looking to take advantage of label-free diagnostics for multiple conditions. For example, researchers at Osaka University have built a diagnostic proof-of-concept platform using a nonlinear optical crystal that emits terahertz waves and uses their closeness to a biological sample in a microchannel to achieve better sensitivity. The system consists of a microfluidic chip that has been used to measure glucose levels in blood at the point of care, but could be applied to many different conditions, according to its developers.