Graphene provides a unique way for sensing local pH level of substances, with important implications in the monitoring of cellular metabolic activities where protonic excretion occurs in suitable conditions [1]. Doping modifications of graphene, induced by the contact of the graphene sheet with different pH solutions were investigated by micro-Raman spectroscopy in order to develop a pH biosensor. A small amount of liquid (of the order of few microliters) was dropped on a graphene surface and the Raman response collected by using He-Ne laser light excitation. Depending on the doping level, the energy of the Raman G mode of graphene significantly changes in the range of 1580-1610 cm-1. The Raman response of graphene exposed to known pH aqueous solutions was investigated in order to provide a calibration curve for the sensor. In the aim to test the developed biosensor with real biological systems, the pH values of cell culture media in different conditions were evaluated. The obtained results suggested that the proposed biosensing scheme could be adopted also for monitoring more complex biological systems as a single cell at the micrometer scale.
[1] G. L. C. Paulus et al. “A graphene-based physiometer array for the analysis of single biological cells” Sci. Reports 4 (2014) 6865.
This is a very insteresting project. Hope I didn´t overlook some information. I got some questions:
1. How homogeneous is the electronic carrier concentration on graphene at the spatial scale at which these experiments are carried out?
2. Wouldn´t it be better to correlate the change of n values (or the percentage of that change) with pH, instead of just n values with pH? I think that the electronic carrier concentration on graphene significantly depends on its synthesis method.
3. How stable are the values from Raman spectroscopic measurements with time?
4. Could you comment on the economic advantages of this technique compared to whatever is commercially available for measuring the pH locally (if any)?
Thanks in advance.
The spatial homogeneity and the time-stability of the pristine graphene are actually critical and relevant aspects of a proper implementation of the sensor considered.
We tested the spatial uniformity of the graphene on an area of 4x4 mm^2 obtaining a standard deviation of 1.36 cm^-1 for the position of the G Raman mode. This value is actually comparable to the spectral resolution of our apparatus, thus the spatial homogeneity of the Raman response of graphene is enough good. However, we observed some significant changes in the Raman response with the time for air-exposed samples that can be originated by air humidity. The sample considered are p-doping and a slight increase with time of the carrier charge density is typically observed.
The effect of this initial doping level on the response of graphene in contact with “acid/alkaline” liquids has been considered in detail by G. L.C. Paulus and coworkers [Sci. Reports 4 (2014) 6865]. The presence of already hole-doped graphene sites reduces the number of sites suitable to be affected by external doping, but, at less for a low level of density of intrinsic doping, the two effects compensate each other. Thus, in the preliminary measurements presented in the communication, we just estimate the doping level variations neglecting the initial doping, even if we agree that a more accurate determination of absolute values of pH level should take in account this aspect.
The possibility to use an optical technique for probe pH level constitutes a unique prerogative of the method considered. Even if Pd-PdO pH microprobes have been proposed ( J.K. Kim, Y.H. Lee, Biotechn. Bioeng. 34 (1989) 131) with spatial resolution of order of micrometers, the Raman spectroscopy has some important advantages: its spatial resolution is limited by the optical one, it is less invasive and Raman response of the sample can give complementary information. An important aspect of the method is that it can be implemented easily by using a standard Raman spectroscopy system.
As concerning the last question, the method considered is mainly addressed to scientific investigations when a high spatial resolution is required. The Raman spectroscopy remains a relatively expensive technique, even if there is a growing interest in this technique and its application in different fields and the Raman equipment is becoming more and more affordable.
