New device monitors levels of inflammation-associated C-reactive protein in real time

Since inflammation is the body’s natural immune response to disease or infection, it increases blood flow in the body and helps the body’s cells defend themselves against viruses, infections, or cell damage. Rapid diagnosis of inflammation is essential for effective treatments, so testing the levels of inflammatory biomarkers in the body is extremely important. Researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences have presented a new point-of-care device that can monitor the levels of C-reactive protein (CRP) produced in the body during inflammation.

The work is published in the journal ACS sensors.

CRP is a molecule that plays an important role in the onset of many diseases of various etiologies. Although CRP has been known and used for many decades to diagnose inflammation in the body, its use as a potential biomarker in the diagnosis of other diseases in the body is relatively new. CRP is a plasma protein produced in the liver and released into the bloodstream in response to the immune system, acting as a biomarker of inflammation and infection. Monitoring its level allows the diagnosis of even serious diseases such as trauma, sepsis, ischemic necrosis and/or malignant tumors.

Since CRP is classified as an acute phase reactant, a single measurement of its level only indicates inflammatory diseases and blood tests must be repeated to determine the effectiveness of a specific therapy. Currently, despite technological advances in biomedicine and medical diagnostics, the techniques used to monitor CRP levels are still far from rapid and real-time measurements.

Recently, researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences, led by Professor Martin Jönsson-Niedziółka, presented a new diagnostic tool that allows for accurate measurement of CRP levels. Moreover, it allows for real-time measurements, thus reducing analysis time and costs. The proposed solution is based on a smartphone-connected point-of-care testing (POCT) device, the size of a credit card, using simple electrochemical biosensors with near-field communication technology integrated into a microfluidic device.

Unlike various biosensors that use large IgG antibodies to capture relevant inflammatory biomarkers, the proposed POCT device offers superior sensitivity and stability, as well as real-time measurements, greatly increasing the potential application in CRP diagnosis. The device equipped with the biosensor can capture and quantify CRP with similar efficiency to conventional and expensive ELISA tests.

The measurements are performed electrochemically using a commercially available portable potentiostat, while CRP capture is performed on the single electrode integrated into the small channels of the microfluidic device. The system also uses near-field communication (NFC) technology, enabling wireless data exchange between the sensors and mobile devices. The results can be displayed on Android smartphones, providing users with an efficient way to collect and analyze electrochemical data in real time.

How does it work? The fluid sample is placed on a small plate of the microfluidic device, which is equipped with a small electrode. Through tiny channels in the device, the sample is gradually introduced into the following parts of the device with an additional solution based on capillary transport without the need for an external pump, which allows for automatic sequential flow. The key role in the demonstrated device is played by anti-CRP nanobodies immobilized on the electrode. Nanobodies are small antibody fragments functioning as an alternative to conventional antibodies used for CRP quantification.

Due to their small size and higher degradation resistance compared to antibodies, nanobodies obtained with the phage display method can be used in different experimental conditions, achieving high antigen binding specificity.

“To immobilize the anti-CRP nanobodies on the electrode, the hydroxyl functional groups of the oxidized electrode were first modified to incorporate aldehyde groups through an oxidation reaction. The anti-CRP nanobodies were then immobilized on the oxidized electrode through an imine bond (C═N). In this way, we obtained the electrode with high specific antigen affinity,” explains Dr. Suchanat Boonkaew, first author of the presented work.

The high surface density of nanobodies on the electrode improves the sensitivity of electrochemical measurements even in the very low concentration range. This allows to achieve an optimal compromise between analytical accuracy in the physiologically relevant concentration range and analysis time.

Dr. Katarzyna Szot-Karpińska adds: “To produce the nanobody-modified electrode, we relied on our experience in biopanning to recover novel CRP-specific nanobodies to be used as active material in the fabricated biosensor.”

The selectivity of the proposed nanobody-based biosensor was tested in various media interfering with the measurements with the presence of biologically active compounds like interleukin-6 (IL-6), fibrinogen, myoglobin, bovine serum albumin (BSA) and human serum albumin (HSA) as well as by exposing the device to different experimental conditions including temperature and humidity changes.

Professor Martin Jönsson-Niedziółka said: “The proposed method was applied to the detection of CRP in human whole blood obtained from anonymous donors. The experimental results showed that the NFC-based system integrated with the continuous flow microfluidic device can correctly quantify CRP in clinically relevant biological samples without the need for pre-treatment procedures, and could therefore be used for the assessment of inflammation, infections caused by bacteria or viruses, and for monitoring the increased risk of heart disease.”

Sooner or later, everyone faces inflammation. Regardless of the etiology, rapid and selective diagnostics that allow real-time monitoring of treatment effectiveness are still far from ideal. Therefore, when rapid and portable point-of-care testing devices are applied in real life, they will undoubtedly revolutionize many areas of medical diagnostics, making life easier for many patients.

The device presented by IPC PAS researchers offers a reliable and cost-effective diagnostic solution that is easy to use and provides rapid results in just a few minutes, without leaving home. It also enables reproducibility in biosensor production and CRP detection, which is a step towards modern and personalized healthcare. Nanobody-based electrodes allow multiple analyses to be performed in real time, accelerating diagnosis and evaluation of treatment effectiveness, enabling immediate therapeutic decisions and improving patient outcomes.