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Repurposing of Waste

Developing sensors from laboratory waste materials.

by Mr Buddhadev Purohit, Dr Kuldeep Mahato, Mr Ashutosh Kumar, and Dr Pranjal Chandra

With the advancement of science and technology, the amount of waste we generate has been increasing exponentially. Thus, making it critical for us to develop newer useful products from the waste, to empower a sustainable future for next generations. Repurposing or recycling of materials not only allows for cost diversion, it is also environmentally friendly.

Findings from the Indian Institute of Technology (IIT), Guwahati, India, researchers have developed a sensor from a laboratory waste material to detect the concentration of hydrogen peroxide (H2O2) in blood samples.1 The fabricated sensor can be made within half an hour and is able to determine the concentration of H2O2 in less than one second.


Metabolic By-Products for Disease Detection

Small biological molecules called enzymes work in the cells to maintain functioning of the human body. These enzymes are able to synthesize complex molecules (anabolism) or breakdown complex molecules to harness energy or create simpler compounds (catabolism). Enzymes belonging to the oxidoreductase class of enzymes, some of which include glucose oxidase, cholesterol oxidase, and glutamate oxidase. During the metabolism, these enzymes are activated to form several by-products, among which H2O2 (Hydrogen Peroxide) is a very crucial one. The effect of H2O2 on the cell varies based on its concentration. At low concentration, it is an indispensable part of our body function involved in several signalling pathways, but at higher concentration it can damage proteins.

At low concentration, H2O2 binds to a protein by sulfenic bond, and directs the signalling process of various cellular functions such as cell proliferation, differentiation, and motility. H2O2 forms a permanent sulfinic and sulfonic bond with the proteins, when present in higher concentrations (more than 100 nM) and alter its functions. Thus, changes in concentration of H2O2 is reported to be associated with various pathological conditions including inflammation, ageing, diabetes, cardiovascular diseases, neurodegenerative disorders, and some cancers in our body.2

Catalases and peroxidases are other classes of enzymes also found in our body that degrade the H2O2 produced to maintain a dynamic steady state concentration. It is very important to maintain a steady concentration of H2O2 for the functioning other associated proteins and enzymes. The precise detection and measurement of H2O2 can be useful to monitor the degree of severity of the associated diseases and help the physician to prescribe medication accordingly.


Detection of Hydrogen Peroxide (H2O2)

Several methods have been developed to detect H2O2 in various samples by using the advanced methods such as fluorescence, colorimetry, chemiluminescence, and spectroscopy. For the detection of H2O2 in biological samples, mostly various colour reagents or chromophore dye are used, that generate a colour complex when came in contact with H2O2. These methods are powerful but suffer from time-consuming protocols and interference by molecules in local environment. There is a need of a detection method that can sense H2O2 with high sensitivity. One of the best ways to measure the H2O2 level in blood is by using horse radish peroxidase enzyme-based methods or by biosensors to get a quick result. But using an enzyme for detection may lead to problems of in stability and shelf life in varied environmental conditions like temperature, pH, and salt concentration. So, for the detection of H2O2, a nonenzymatic sensor probe without any label is the most efficient way.3


Biosensors for Detection of Hydrogen Peroxide (H2O2)

Biosensors are new age diagnostic instrument used for the detection of the analytes. These analytical devices comprised of a recognition unit, one transducer system, and one processor unit. When the recognition unit detects a specific molecule in the sample, the transducer and processor will analyse the signal and convert it, often in form of digital image that can used for further quantitative analysis.4 Among many types of biosensors, electrochemical biosensors are most commonly used due to their fast and accurate detection.5 The developed sensor can electrochemically detect the concentration of H2O2 in blood samples. The principle behind the developed electrochemical sensor is that it can catalyse the reduction of H2O2 at a particular applied potential and simultaneously detect the thus generated electrons. The increased flow of electron can be correlated with the presence of higher concentration of H2O2. To develop the sensor, a special class of nanoparticles known as metallic dendrites are directly grown over the indium tin oxide sensing electrode. The metallic dendrites are a class of nanomaterials that look like a fern or tree pattern with primary, secondary, and tertiary branches.6 The metallic dendrites are electrochemically very active material with a large surface area which can catalyse various reactions based on its material property and geometric orientation of the active nanoparticles.


Sputtering Gold Dendritic Electrode

Serendipitously, they came across the higher activity of the dendrites after gold sputtering, which was done for scanning electron microscopy (SEM) imaging. In biological research, the imaging of samples by SEM is an inseparable method, to look at the morphological property with finer details. In SEM imaging, a very high voltage of electron is bombarded over the sample for high resolution images, but it has the potential to damage the sample. To withstand the high energy beams, a very thin layer of gold nanoparticles is sputtered over the sample. The developed sensor electrode was sputtered with gold nanoparticles prior to SEM imaging. After the imaging, the SEM images of the materials are kept for further use but the sputtered electrode with material was usually thrown away as it serves no purpose.

However, it was found that the sputtered gold dendritic electrode was seen to be brighter than the non-sputtered one. Curiously, testing its electrochemical properties by running various experiments it was found that the conductivity had increased. Upon checking the catalytic activity and found that it had the ability to catalyse the H2O2 reduction reaction with twice the efficiency than a non-sputtered electrode. The enhanced peroxidase activity might be due to the effect of bombarded nanoparticle to create defects on the dendritic electrode surface, thus leading to an increase in the active surface area.


Dynamic Range and Accuracy

The developed sensor reported a wider dynamic range of 1 x 10-5 to 1 x 10-12 M, and a limit of detection of 9.8 x 10-13 M. The concentration of blood H2O2 in healthy person (1 nM) and in the case of diseased person (100-1000 nM) this falls within the detection range of the fabricated chip sensor signifying its potential. The sensor is capable of detecting H2O2 even in the presence of high concentration co-existing molecules like glucose, urea, uric acid, glycine, and alanine. The sensor was able to detect the presence of H2O2 in blood samples with 96 percen accuracy. We further tested that the error in generated signal by multiple probe was minimal and the sensor was stable up to a period of 12 weeks with negligible loss of its electro-catalytic activity. The sensor chip developed from trash is found to be more efficient than the recently reported peroxidase sensors.

This sensor can be used for the detection of blood H2O2 levels in the hospital and other clinical settings. The quick and sensitive detection can be useful to determine the level of disease progression and assist further medication. Not only blood, the system can be used for the peroxide detection in other sample such as milk, oil and food products with proper modification and optimisation of the sensor chip. Especially in milk, H2O2 is mixed to prolong the shelf life of milk products, which causes negative health effects on the consumer. The FDA guideline for milk H2O2 concentration falls within the range of our sensor chip. As the sensor chip is devoid of any biological molecules, it will be less affected by the presence of various inhibitors or extreme physical parameters. Further, a handheld kit with improved analytical performances can be developed in the future to assist the sensing of H2O2 onsite for clinical as well as industrial applications. [APBN]

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