Periodic Reporting for period 1 - SOON (Sustainable Nitrite Biosensor for Food Safety monitoring)
Berichtszeitraum: 2020-05-04 bis 2022-05-03
Nitrites are commonly used as corrosion inhibitors and in numerous medical and industrial processes. The food manufacturers employ it to preserve the food and to prevent the growth of harmful microorganisms, in particular Clostridium botulinum, a motile bacterium with the ability to produce the neurotoxin botulinum. Additionally, nitrite is the intermediate specie of the nitrogen cycle and can interact with amines to form carcinogenic nitrosamine compounds. Nitrites and nitrites metabolic compounds such as nitric oxide, have raised concern over potential adverse health effects and its use is strictly regulated [Commission Regulation (EU) No. 601/2014, No.1129/2011]. For example, the amount of nitrite allowed to be used as a food additive in cured meat is currently 150 mg kg−1 (expressed as NaNO2), except for somewhat higher levels in some traditional cured products however control of specific food additives such as nitrites, and is still a problem that must be controlled.
The aim of SOON project was to evaluate and demonstrate the feasibility of producing sustainable biosensors that can be applied for on-site nitrite detection.
The aim of SOON project was to evaluate and demonstrate the feasibility of producing sustainable biosensors that can be applied for on-site nitrite detection.
Important achievements that have been (or will be) exploited at BIOLAN have been obtained. From the industrial perspective, the most important one is the development of a novel device comprising an amperometric biosensor for the detection of nitrite without the needed to use any oxygen scavenging. This strategy has been successfully implemented at BIOLAN. The implementation of SOON, by the ER and the hosting institution, will give for the first time to the food industry, a truly portable, long-term stable and reproducible enzyme-based electrochemical biosensor for nitrite detection.
Optimization and validation of the new nitrite SPE biosensor results in an improved biosensor platform. A market study is undergoing to evaluate the possibility of the commercialization of nitrite SPE biosensors. Using the developed protocol at least 6 times, more biosensors are produced in a faster manner. The linear range of the new biosensor goes from 1 to 60 mg L-1 of NO2- with a correlation coefficient of 0.9987 and a slope of 0.943 ± 0.012. Different real samples were analysed and a high correlation between the theoretical and experimental values was found, evidencing the usefulness of the biosensor for the determination of NO2- in real samples.
The ER also participated in the development and improvement of other enzyme based biosensors:
• A new xylose biosensor was developed and validated for the determination of lactose intolerance with excellent results. The use of the biosensor platform allows the determination of hipolactasia in 30 s in contrast with the gold standard method that take at least 30 min. Using the developed protocol at least 5 times more biosensors are produced in a faster manner.
• A proof of concept Nitrate and Nitrite bienzymatic biosensor was developed for the determination of these analytes in meat samples with good results and without the needed of derivatisation of the nitrite to nitrate for the determination of both of them. The bienzymatic biosensor shows excellent analytical characteristic and was used for the determination of the analytes in a quality control material supplied for FAPAS.
Optimization and validation of the new nitrite SPE biosensor results in an improved biosensor platform. A market study is undergoing to evaluate the possibility of the commercialization of nitrite SPE biosensors. Using the developed protocol at least 6 times, more biosensors are produced in a faster manner. The linear range of the new biosensor goes from 1 to 60 mg L-1 of NO2- with a correlation coefficient of 0.9987 and a slope of 0.943 ± 0.012. Different real samples were analysed and a high correlation between the theoretical and experimental values was found, evidencing the usefulness of the biosensor for the determination of NO2- in real samples.
The ER also participated in the development and improvement of other enzyme based biosensors:
• A new xylose biosensor was developed and validated for the determination of lactose intolerance with excellent results. The use of the biosensor platform allows the determination of hipolactasia in 30 s in contrast with the gold standard method that take at least 30 min. Using the developed protocol at least 5 times more biosensors are produced in a faster manner.
• A proof of concept Nitrate and Nitrite bienzymatic biosensor was developed for the determination of these analytes in meat samples with good results and without the needed of derivatisation of the nitrite to nitrate for the determination of both of them. The bienzymatic biosensor shows excellent analytical characteristic and was used for the determination of the analytes in a quality control material supplied for FAPAS.
Strategic studies and market research were made to determine the risks, opportunities and possible applicability associated with the design, process and production of all the components of the nitrite SPE biosensor.
A mass production manufacturing of the NO2- SPE biosensor (n=1000) electrodes were produced using an automated precision liquid dispensing instrument (Biodot) with the aim to facilitate a high volume production of biosensors with similar characteristics.
Enzyme production process is being fine-tuned at BIOLAN. The projection is that, by early 2023, it will be fully developed and can be used to prepare biosensors which would increase by at least 6 times the production yield, considerably reducing the time and effort needed for the development of the biosensors, and consequently, decreasing the cost of manufacturing. Besides, the developed protocol is highly reproducible and less complex than the previously used in the development of this kind of biosensors, a fact that ensures a uniform and high quality product.
The developed Nitrite SPE biosensors have been coupled to the BIO7000 measurement devices developed by BIOLAN. The portable and user-friendly equipment that currently markets BIOLAN is a miniaturised potentiostat for reading test strips. The equipment, powered by a USB rechargeable battery, consists of Bluetooth and USB communications, includes a button with ON/OFF function, and have an IP65 sealing, except in the entrance area of the reactive strips. In addition, the equipment has a detection system for the presence of strips to ensure its correct introduction. And it also has the possibility of reading two working electrodes at the same time, which are integrated in the same strip. Differences were not observed when the nitrite SPE biosensors were used in the BIO7000 device developed at BIOLAN when compared with a commercial potentiostat (Palmsens).
A mass production manufacturing of the NO2- SPE biosensor (n=1000) electrodes were produced using an automated precision liquid dispensing instrument (Biodot) with the aim to facilitate a high volume production of biosensors with similar characteristics.
Enzyme production process is being fine-tuned at BIOLAN. The projection is that, by early 2023, it will be fully developed and can be used to prepare biosensors which would increase by at least 6 times the production yield, considerably reducing the time and effort needed for the development of the biosensors, and consequently, decreasing the cost of manufacturing. Besides, the developed protocol is highly reproducible and less complex than the previously used in the development of this kind of biosensors, a fact that ensures a uniform and high quality product.
The developed Nitrite SPE biosensors have been coupled to the BIO7000 measurement devices developed by BIOLAN. The portable and user-friendly equipment that currently markets BIOLAN is a miniaturised potentiostat for reading test strips. The equipment, powered by a USB rechargeable battery, consists of Bluetooth and USB communications, includes a button with ON/OFF function, and have an IP65 sealing, except in the entrance area of the reactive strips. In addition, the equipment has a detection system for the presence of strips to ensure its correct introduction. And it also has the possibility of reading two working electrodes at the same time, which are integrated in the same strip. Differences were not observed when the nitrite SPE biosensors were used in the BIO7000 device developed at BIOLAN when compared with a commercial potentiostat (Palmsens).