A Raman-based biosensor for measuring extracellular hydrogen peroxide concentrations

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Figure 1. (a) Explanatory sketch of the plasma membrane anchored nanosensor. The compounds used for gold nanoparticles (AuNP) conjugation are the H2O2-sensitive 4-mercaptophenylboronic pinacol ester (4MPBE) and Biotin-HPDP. Biotinylation of the plasma membrane protein ectodomains is obtained using NHS-Biotin. The anchoring of the conjugated AuNP and NHS-Biotin is given by Streptavidin reacting with the two biotin moieties. (b) AFM analysis was performed on A549 lung cancer cells after nanosensor anchoring and fixation. High resolution AFM images confirmed the presence of the nanosensor, which is in contact with the cell surface and is capable of detecting endogenous H2O2 in a very shallow region (i.e., 90 nm) of the extracellular fluid in contact with the plasma membrane.

Several processes in the human body are regulated by biochemical reactions involving hydrogen peroxide (H₂O₂). Although it can act as a “secondary messenger”, relaying or amplifying certain signals between cells, H₂O₂ is generally toxic because of its oxidant character. The oxidising property of H₂O₂ is of potential therapeutic relevance for cancer, though: deliberately causing tumour cells to increase their H₂O₂ concentration would be a way to destroy them. In light of this, but also for monitoring pathologies associated with H₂O₂ overproduction, it is crucial to have a means to reliably quantify H₂O₂ concentrations in the extracellular environment. Now, Leonardo Puppulin from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University and colleagues have developed a sensor for measuring concentrations of H₂O₂ in the vicinity of cell membranes, with nm resolution.

The biosensor consists of a gold nanoparticle with organic molecules attached to it. The whole cluster is designed so that it anchors easily to the outside of a cell’s membrane, which is exactly where the H₂O₂ molecules to be detected are. As attachment molecules, the scientists used a compound called 4MPBE, known to have a strong Raman scattering response. When a 4MPBE molecule reacts with a H₂O₂ molecule, its Raman spectrum changes. Based on this principle, by comparing Raman spectra, Puppulin and colleagues were able to obtain an estimate of the H₂O₂ concentration near the biosensor.

Figure 2. (a) Surface enhanced Raman spectroscopy was used to detect and quantify the H₂O₂-induced modification of 4MPBE molecules assembled on the gold surface of the nanosensor anchored to the cells. The intensity of the Raman band at 998 cm^–1 depends on H₂O₂ concentration, while the band at 1074 cm^–1 did not show modification and it can be used for normalisation of the spectral lines. (b) Results of the nanosensor calibration. The ratio of the intensity at 1074 cm^–1 to the intensity at 998 cm^–1 showed linear dependence with respect to H₂O₂ concentration. (c) Example of SERS hyperspectral map of extracellular endogenous H₂O₂ collected from the surface of the A549 cell shown in the bright-field image. The SERS spectra shown in (a) were collected from the locations A and B.

After developing a calibration procedure for their nanosensor, the scientists were able to produce a concentration map with a resolution of about 700 nm for lung cancer cell samples. Finally, they also succeeded in extending their technique to obtain measurements of the H₂O₂ concentration variation across cell membranes.

Figure 3. (a) Explanatory schematic of the extracellular production of H₂O₂ by NOX complex, the consequent absorption through Aquaporin channel and the reaction with intracellular peroxiredoxin. At steady-state condition, the concentration ratio of extracellular H₂O₂ to intracellular H₂O₂ can be estimated by the ratio of the rate constant of H₂O₂ reduction by peroxiredoxin (kprx) to the rate constant of H₂O₂ absorption through the plasma membrane (k_abs). (b–c) Typical results of redox biology experiments with fitting lines from which we measured k_abs and k_prx, respectively. (d) Mean surface extracellular [H₂O₂]_e and the typical maximum of [H₂O₂]_e measured on A549 cells using the newly designed nanosensor. (e) Mean intracellular [H₂O₂]_i and the typical maximum of [H₂O₂]_i estimated on A549 cells according to the model reported in (a) and using the results from (b–d).

Puppulin and colleagues conclude that their “novel approach may be useful for the study of actual H₂O₂ concentrations involved in cell proliferation or death, which are fundamental to fully elucidate physiological processes and to design new therapeutic strategies.”