Monitoring the level of copper in the environment is important, as both the redundancy and deficiency of this transition metal can have severe effects on living organisms. Copper helps the body make red blood cells and keeps nerve cells and the immune system healthy. It also helps form collagen, which is a key part of bones and connective tissue. Thus, copper deficiency causes hematological and neurological disorders. Besides, copper toxicity has been known to cause Alzheimer’s diseases as well as inflammatory disorders in humans.
According to the US Environmental Protection Agency (EPA), the acceptable level for copper concentration in drinking water should be lower than 1.3ppm. There are many advanced standard techniques available, such as inductively coupled plasma mass spectroscopy (ICP-MS), chemiluminescence, etc., for assessment of copper concentrations. However, heavy instrumentation, requirement of skilled personnel, long sample processing time, and high costs limit the field applications of these methods. Recently, several fluorescence turn-on or turn-off based organic dye molecules have been reported which can detect very low concentrations of copper ion, both in vitro and in vivo. Moreover, electrochemical methods have gained prevalence for detection of copper at low concentrations.
Nevertheless, most of these approaches are challenged by the presence of impurities and pH of the sample solution, and a few of them work only in pure organic solvents. Therefore, it is difficult to estimate copper concentration in natural sources. Thus, we embarked on developing a portable label-free optical biosensor device that can detect a wide range concentration of copper ions in human blood serum as well as environmental samples.
We probed a unique detection method of copper ions using denatured human immunoglobulin (HIgG) as the receptor molecule. It is known that Cu (II) is a redox active transition metal capable of oxidative reactions. It has binding affinity to different amino acids of a protein. In particular, the side chains of Histidine (His) and Cysteine (Cys) are known to form strong complexes with copper. Cu (II) reacts with free sulfhydryl (-SH) groups available in the hinge region of IgG to form Cu (II)-SH complexes which result in oxidation of thiol to its radical form SR* and Cu (II) gets reduced to Cu (I). Later, both radicals react to form the corresponding disulfide, and Cu(I) gets oxidized to Cu(II).
In the native folded form, all the available binding residues of HIgG may not be exposed. However, denaturation of immunoglobulin can break the disulfide bonds of the IgG structure, thereby increasing the free thiol (-SH) sites as well as the amount of exposed histidine for coordination with the copper. Based on this property, we hypothesized that the redox reactions of copper ions with denatured immunoglobulin G (IgG) on the polyaniline (PAni) surfaces may have a significant effect on the molecular conformation of the polyaniline backbone, which may result in spectroscopic changes in absorbance. In this work, three different methods were used to denature the IgG, i.e., heat treatment, chemical cleavage, and UV-ray exposure, and their copper binding ability were estimated separately.
Denatured antibody immunoglobulin G (IgG) was immobilized on polyaniline (PAni), which in turn is the coating on the core of an optical fiber. This sensing relies on changes in evanescent wave absorbance in the presence of the analyte. Interaction of copper ions with denatured IgG on PAni polymer surface induces radical reactions which significantly changes the EW absorption properties of the polymer. We utilized this feature for the detection of copper ions in environmental samples. UV-exposed denatured IgG showed extremely high copper ion sensitivity compared to native IgG because of the high availability of copper binding sites. The sensor showed excellent selectivity for Cu (II) ions in the presence of all other divalent metal ions and works for a wide range from 10 µM to 10fM with a good correlation coefficient (R2=0.98).
The sensor was tested in natural water bodies, such as lake water and marine water, to determine unknown concentrations of copper ions, and the recovery results were within 90% to 115%, indicating reasonable accuracy. The sensing ability of the sensor was further extended toward successful detection of copper ions in blood serum and soil samples, manifesting its potential for a wide scope of field study. We further integrated the sensor with a miniaturized, hand-held instrumentation platform to develop a portable device for field application. Overall, this study demonstrates the potential applicability of PAni coated fiber-optic sensor in conjunction with IgG protein for the determination of copper in real samples by means of a portable, accurate, and field-deployable device.
These findings are described in the article entitled Hand-held optical sensor using denatured antibody coated electro-active polymer for ultra-trace detection of copper in blood serum and environmental samples, recently published in the journal Biosensors and Bioelectronics. This work was conducted by Sutapa Chandra, Arvind Dhawangale, and Soumyo Mukherji from the Indian Institute of Technology Bombay.