Detecting Inflamatory Markers from Macrophages Using Micropatterned Electrochemical Biosensors

Macrophages are one type of immune cell that is responsible for the primary contact with the pathogen, leading to an inflammatory process by the release of cytokines and reactive oxygen species (ROS).  ROS be classified as chemical species formed by incomplete reduction of oxygen, which includes the superoxide anion (O₂˙), hydrogen peroxide (H₂O₂), and hydroxyl radical (˙OH). ROS compounds are naturally unstable and, upon degradation can form more stable compounds such as H₂O₂. Therefore, the release of H₂O₂ is an importante analyte for monitoring the ROS production by immune cells. 

Inflammatory processes and the H₂O₂ production play a significant role on several pathologies, including: diabetes, cancer, alcoholic liver disease, Alzheimer’s progression, Parkinson’s and tissue fibrosis.  The inflammatory signals are required in normal conditions to combat pathogens associated with injuries or infections, but may have harmful pathophysiological consequences if unregulated. 

In this work, we develop a integrated electrochemical detection system, with H₂O₂ biosensors, and the cell culture integrated in microfluidic devices. For this, we used a different strategy using biomaterial micropatterning which enable the efficient placement of macrophages surrounding the proximity of H₂O₂ biosensors and ensure that the sensing electrodes were not fouled.  To accomplish this, we utilized gold electrode arrays in quartz/chrome plates (75mm x 25mm) using standard microfabrication process and integrate an enzyme-containing hydrogel microstructures.            

In order to integrate the Horseradish oxidase (HRP) into PEG-containing solution, we used an enzyme concentration of 10 mg/mL. In parallel, the prepolymer solution was prepared by adding 2% (v/v) photoinitiator (2-hydroxy-2-methyl-propiophenone) to 1 mL of PEG-diacrylate (PEG-DA). The HRP-PEG hydrogel prepolymer solutions were prepared by adding 5 µL, 10 µL, 20 µL, and 40 µL of the enzyme solution mixed with glutaraldehyde into 50 µL of PEG-DA.

The procedure for patterning PEG-enzyme microestuctures on electrodes was performed by exposure to UV light, where the PEG-based prepolymer solution was spin-coated at 800 rpm for 4 sec onto glass slides containing Au electrode patterns. A photomask was fabricated with the same electrode pattern and then exposed to UV light at 65 mW/cm² for 10 sec to convert liquid prepolymer into cross-linked hydrogel.  The surfaces were cleaned in DI water for 3 min to remove unpolymerized PEG solution.  Enzyme carrying hydrogel microstructures were made larger than Au electrodes (600 mm and 300 mm diameter for hydrogel and Au features respectively).

After fabricating enzyme-based hydrogel/Au electrodes and outfitting these electrodes with microfluidic channels, we characterized the response of these microdevices to known concentrations of H₂O₂.  It should be noted that H₂O₂ can be reduced or oxidized at negatively or positively poised electrodes.

We evaluate the optimal potential for performing H₂O₂ detection.  In these experiments, hydrogel/Au electrodes with and without HRP were poised at potentials ranging from - 0.3 to 0.3V vs. Ag/AgCl and were challenged with 200 µM of H₂O₂.  The results reveals several insights into signal vs. applied potential relationship: 1) hydrogel modified electrodes exhibited higher current density at reductive (negative) voltages, 2) HRP-containing hydrogel/Au electrodes were ~ 60 times more sensitive to H₂O₂ compared to gel-coated electrodes without enzyme at working potential -0.2 V and 3) the presence of HRP in the hydrogel did not contribute to improved sensitivity at oxidative (positive) potentials. 

And we also evaluated the selectivity of sensor, since is possible to an electrochemical biosensors working under physiological conditions have your real signal masked by interference from nonspecific compounds undergoing electrochemical reactions at the sensing electrode.  Therefore, our biosensor was challenged with the analyte of interest (H₂O₂) as well as with 5 most common interfering species that are generally used to test the H₂O₂ biosensor selectivity prior cell experiments: AA, UA, DOPAC, SO₃^2- and O₂.  Interfering substances had minimal effects compared with sensor response to 100 µM H₂O₂.

PEG hydrogel micropatterning was employed to both fabricate HRP-containing electrochemical biosensors and to define living cell/biosensor interface. A microfluidic chamber served the purpose of a three-electrode electrochemical cell with HRP/hydrogel/Au electrodes serving as working electrodes. A microdevice with integrated H₂O₂-sensing electrodes had sensitivity of 28 mA/cm² mM and lower limit of detection of 2 µM.

These results indicate a real applicability of the proposed biosensor. Featuring a representative experiment, where it was possible to verify the liberation of H₂O₂ from macrophages in real time and with refined ranges.

ACKNOWLEDGMENT: The financial support for this study was provided by CNPq.