The field of synthetic biology has exploded over the past decade, having a major influence on fields such as metabolic engineering, protein engineering, digital biology, and whole-genome engineering.

Biosensors & Biochips Technologies: Contribution to the Future Sustainable Life

The field of synthetic biology has exploded over the past decade, having a major influence on fields such as metabolic engineering, protein engineering, digital biology, and whole-genome engineering. In the framework of iterative “design-build-test” development cycles, a significant portion of synthetic biology innovation has taken place. In the field of synthetic biology, progress can be associated with innovations in each of the processes of “design”, “build” and “test”. For example, there has been a major push to standardize elements within synthetic biology, with significant attention being paid to modularity and “plug and play” components. This modularization, along with the accelerated progress in systems biology, has allowed the “design” stage to become less time-consuming and less reliant on advanced knowledge. In recent years, the cost of DNA sequencing and synthesis has also decreased dramatically, allowing large constructs to be synthesized cheaply. In the ‘build’ phase, this has facilitated a rapid improvement, helping researchers to investigate a larger percentage of the space of the biological solution. Finally, within the synthetic biology “test” phase, high-throughput screening has also become a focal point. The increased “design” and “build” potential has contributed to an increased demand for success in the assessment of the plethora of new designs. In turn, this was done by incorporating robots and high-throughput analytics into the laboratory setting, in which new models can be evaluated to a level that is not achievable for human researchers.

Biosensors represent a groundbreaking emerging technology for high-throughput screening that can be implemented. Most precisely, they are classified as an analytical tool consisting of biological components used to detect and generate a signal for the presence of a target ligand. Synthetic biology is at the forefront of biosensors, both as a tool for high-throughput screening, but also as the direct result of developments within the field of synthetic biology itself. In addition, because of the unparalleled specificity and sensitivity that biological parts provide relative to conventional analytical methods, biosensors have gained expanded interest as alternatives to traditional analytics.

The design and construction of biosensors is a multidisciplinary endeavour and can include expertise in areas such as protein engineering, molecular biology, affinity chemistry, molecular dynamics of nucleic acid, materials sciences, and nanotechnology. Biosensors interface with a target ligand at their most simple stage, undergo some type of modification, and output a signal. There is a great variety of potential configurations in all the parts of this process. Target ligands range from single atoms such as calcium, to entire proteins such as thrombin, all the way through. Processes as varied as enzymatic activity, fluorescence, electrical current generation, and transcriptional activity include output signals. The mechanisms that transduce ligand recognition into functional signals are just as diverse.

In the field of analytics, biosensors represent a significant step forward. In order to move analytics away from purely physics- or chemistry-based frameworks, the integration of biological components in sensory diagnostics has begun. This has allowed analytical functions that are not well adapted to conventional methods to conduct a vast diversity and specificity of biological components. The theoretical and demonstrated biosensor applications cover a significant range of human society and activity. Biosensor applications are grouped into three broad categories, depending on their measurement scale.

• Group Diagnostics: Environmental, Agricultural, and Industrial Applications
• Point-of-Use Diagnostics: Medical, and Security Applications
• Single-Cell Diagnostics: Metabolic Engineering, and Synthetic Biology Applications

The current food production faces immense challenges from the increasing human population, the maintenance of clean resources and food quality, and the protection of the environment and climate. Food sustainability is mainly a cooperative effort that results in the development of technology funded by both governments and companies. Several attempts have been supported to overcome challenges and improve the drivers in food production. Via their applications, biosensors and biosensing technologies are widely used to solve the major challenges of food production and its sustainability. As a result, there is a rising need for biosensing technology in the area of food sustainability. A technological system combining several technologies is defined by microfluidics. Nanomaterials, with its biosensing technology, is known to be the most innovative tool strongly associated with world populations in dealing with health, energy, and environmental issues. The need for point of care (POC) technology in this area focuses on analytical tools that are fast, simple, precise, compact, and low-cost.

For our existence and lives, food with its production industry is essential; and its sustainability is essential in continuous human growth on the planet. Current food production is facing immense difficulties from increasing human population, maintaining clean resources and food quality, and protecting environment and climate. Some of these issues stem from food production itself; others stem from other food production-related industries. Food recalls, for example, trigger major damage to food brands’ credibility and prestige, with an estimate of $15 million per incident over the last few years. 48 million sick cases are responsible for 3000 fatalities annually due to foodborne illnesses.

Food safety is largely a cooperative effort arising from both governments and companies in technology development. In order to pose new challenges in food safety issues, information technologies such as blockchain technology can accelerate communication between food quality, media and consumers. Five challenges can be summarized as the main challenges in the sustainability of food production: the production challenge of food safety and security; the quality challenge of food diversity and quality; the economic challenge in the leading food system, including its packaging and supply chain; the environmental challenge, including the processing of food waste; and the engineering challenge in the creation and generation of novel food.

Basically, a biosensor is an analytical instrument used to measure a sample’s molecule of interest (target). In general, a bio-recognition factor (aptamer, antibody, enzyme, etc.) that is unique to the target is used. A physiochemical or biological signal is elicited by molecular recognition events between the recognition element and the target compound, which is transformed into a measurable quantity by the transducer. Signals are shown in either optical (colorimetric, fluorescence, chemiluminescence and plasmon surface resonance) or electrical (voltammetry, impedance and capacitance) or any other chosen format (Figure 3).

Figure 3. Classification of biosensors based on transducer and bio-recognition elements used in food analysis

As one of the primary objectives of food analysis, food safety is a major health issue in both animal and human lives. The advancement of food safety analytical technology means that it thrives in line with the rising interest in and emphasis on food supply safety issues. In food safety analysis, traditional approaches are labour-intensive, time-consuming, and need trained technicians. The application of microfluidics in food safety analysis provides fresh insight about how to detect foodborne toxins, allergens, pathogens, hazardous substances, heavy metals, and other contaminants effectively and rapidly. Microfluidics’ features, such as it miniaturize-capability, compact and reducible quantities of samples and reagents, make it a perfect technology for the development of food sustainability. Complex food matrix preparation and difficult manufacturing steps are the current challenges in the application of microfluidics to food sustainability. These challenges can be addressed by leveraging physical properties dependent on specific test targets, designing complex real food analysis microfluidic platforms, and incorporating into microfluidic systems biomolecules such as food proteins and DNA.

Nanomaterials in biosensing technology

With its biosensing technology, nanomaterials are the most promising tool in dealing with health, energy and environmental problems associated with population in the world. Particles smaller than 100 nm in at least one size dimension are known as nanomaterials. These nanomaterials are biocomposite polymers based on metal, metal oxide and carbon, and different types of nanoparticles have been established, such as magnetic iron, aluminum, gold, silver, copper, silica, zinc, zinc oxide, cerium oxide and titanium dioxide nanoparticles, and single/multiple walled carbon nanotubes (CNTs). Nanotechnology and its agricultural development have been greatly extended in different fields. These fields include food production, crop protection, detection of pathogens and toxins, purification of water, food packaging, disposal of wastewater, and environmental remediation. Improving the productivity and performance of applications is the priority of these agricultural fields.

In the field of food safety and protection, biosensing technologies have been developed for nutrient and quality detection, detection of pathogens and detection of toxins, as listed below.

Nutrient and quality detection

Food protection measures can be split down into two categories: post-harvest loss and food biosecurity. Food biosecurity means food contamination and degradation, which is addressed in the later sections, by environmental, political, unfair economic gain, warfare, or exacting revenge. Post-harvest loss, on the other hand, suggests the nutrients and edible conditions in food that need to be maintained between the harvest period and the moment of consumption by technologies. Since time differs from minutes to years, in maintaining and reducing losses, technologies focusing on reducing post-harvest losses are important.

To maintain food quality and to avoid post-harvest losses, new technology such as biosensing can be used. Biosensors have been developed, for example, to detect and analyse quantities of sweeteners in foods that can be used to detect both natural and artificial sweeteners. Sweeteners are widely used in food production and processing, but they have recently been identified in humans as causing health problems. A multi-channel biosensor has been developed to use electro-physiological sensing from taste epithelia to detect and analyse both natural and artificial sweeteners. To detect long-term signals from sucrose, glucose, cyclamate, and saccharin, respectively, the signals are studied through spatiotemporal techniques. The biosensor can distinguish between different concentrations with dose-dependent increased responses of the taste epithelium from different sweeteners. It can also distinguish between two natural sweeteners: sucrose and glucose, with two signal patterns. For glucose, the detection range is 50-150 mM, and for saccharin, 5-15 mM.

Detection of pathogens

Due to their reduced format, biosensors targeting pathogen detection such as bacteria (Table 1) and fungi (Table 2) started more than two decades ago; one device to address multiple problems, and a multi-panel signal detection. The ligand motif is a crucial element in the biosensor design for pathogen detection since it determines the sensitivity and efficiency of the device. The aim is to establish a fast, specific, and sensitive platform to detect in food samples the presence or absence of pathogens. It has been discovered that there is no ideal ligand, and various ligands have different advantages. The combination of bioreceptors to detect a large variety of microbes in different samples poses current challenges in pathogen biosensor detection; new synthetic ligand designs such as aptamers, small molecules, and peptides; and the incorporation of different ligands into a portable device to achieve rapid, effective, and low-cost detection.

Detection of toxins

The mainstream of development in food safety is electrochemical biosensors for rapid detection and assessment of food toxins. Numerous platforms have been developed to allow customized and individualized devices to meet particular environmental and organizational requirements and to reach the nM to fM detection limit levels. For example, to encourage unique binding profiles, bioreceptor arrays address individual electrodes functionalized with different bioreceptors with binding targets. In addition to electrochemical biosensing, toxin and chemical detection in food production have been applied to other biosensors such as optic and piezoelectric sensing (Figure 4). In order to sense toxins, fluorescent nanoparticles have been produced in foods and bodies, including on-surface, inter- and intra-cellular foods.

Figure 4. Predominant food contaminants and the target analytes in the food manufacturing industries

Toxin extraction from complicated food samples is one of the main obstacles in creating a fully automated toxin detector. To automatically assess their harmful levels from food and water samples, potential systems are expected to extract, process, and measure toxins. In identifying, discriminating, and quantifying chemical toxins in food matrices, sophisticated separation strategies have been coupled with SERS. In addition, even though they are typically in lower amounts, chemical contaminants from food processing can be a challenge. Lower stability, selectivity and sensitivity are another challenge in food toxin detection, where MIPs can be a solution to provide stable and low-cost alternatives.

Heavy metals like Ag⁺, As³⁺, Cd²⁺, Hg²⁺, Pb²⁺, and Zn²⁺ are known as chemical pollutants that form stable states of oxidation and interfere with metabolic pathways, resulting in health problems. Aptamer and DNA-based biosensors can detect heavy metals at both nanoscale and very large-scale levels, which are appropriate for food safety screening and monitoring. In order to detect arsenate in food, a heavy metal detecting biosensor is based on genetically modified bacterial cells and a green, fluorescent signal amplifier. With a detection range of 5-140 μg/L of arsenic, its arsenic detection lasts just one hour and can be integrated with optical power output for its future biosensing optical fibre. Other biosensing technologies like aptamers, nanoparticles and graphene electrodes have been successfully applied to the identification and evaluation of arsenic, with the potential to be produced as fast, simple, easy-to-use, and low-cost devices.

Nanotechnology has been adapted to two separate fields of agri-food pesticides: as a pesticide delivery vector for pesticide management and as a trace-amount detector for pesticides. In the first field, nanoparticles are able to slowly modify pesticides to target insect pests, which helps prevent groundwater and topsoil pollution, reduce pesticide levels and improve efficiency. In the second field, bio- or biomimetic-based nanotechnology, like antibodies, enzymes, aptamers, and MIP-like macromolecules, improves stability, selectivity, sensitivity, and speed of detection. Furthermore, bacterial, fungal, algal, and mammalian cells are all cell-based biosensors used in pesticide and herbicide detection, helping to establish fast, reliable, real-time, and cost-effective tools for decontamination procedures and preventive casualty damage.

Carcinogens, odorants, and marine contaminants are other toxins that are significant in food production. Carcinogens are a complex group of trace amount of toxins, like pesticides, heavy metals, mycotoxins, and acrylamide, in which the difficulty of identifying trace-amounts is a challenge; and imprinted aptamers, nanotechnology, and biosensing are optimistic for promising future use. Sensitive and soluble molecules effective in odour detection for olfactory animal systems are odorant binding proteins. A nanosensor combining localized SPR and small odorant binding proteins from honeybees has been established in which the detection range is 10 nM – 1 mM using a quantitative array of nanocups. To monitor and preserve a stable environment for marine food systems, marine contaminant detection is used. Finally, through their sensitive detection capabilities, miniaturized devices, wireless communication, and small-scale networks, biosensors can be applied to marine food safety to be established as advanced analytical and monitoring tools.

Another development kit for food safety biosensors focuses on the detection of genetically modified organisms (GMOs) in food products. Since the 1990s, GMOs in all fields of agricultural products have been considered a biotechnology revolution. To present, more than 45 percent of the world’s soybeans, 40 percent of corn, and 50 percent of cotton are GM products; and GM is also used in livestock. Recent research, however, indicates that GMO products can affect human and animal bodies through gastrointestinal problems, antibiotic resistance, allergenicity, diversity of farm products degradation, and undesired gene flow to other species. Biosensors are designed to measure GMOs in foods and feeds using isothermal DNA amplification and fast detection signal detection to identify GM genes. Detecting unidentified DNA genes that can be resolved by high-throughput technology like the combination of biosensing and arrays, and the development of databases of GMO genes are the key challenges in GMO detection.

Biosensors & Biochips for environmental monitoring

Due to the strong connection between environmental pollution and human health/ socioeconomic progress, environmental monitoring has become one of the priorities on a European and global scale. Biosensors have been commonly used as cost-effective, rapid, in situ, and real-time analytical techniques in this field. The recent development of biosensors with new transduction materials obtained from nanotechnology and for multiplexed pollutant detection, involving multidisciplinary experts, explains the need for compact, fast, and smart biosensing devices. Several recent developments exist in the monitoring of air, water, and soil contaminants by biosensors under real conditions, like pesticides, highly toxic components and small organic molecules, including toxins and endocrine disrupting chemicals.

Biosensors used in environmental monitoring can be categorized as optical (including optical fibre and surface plasmon resonance biosensors), electrochemical (including amperometric and impedance biosensors) and piezoelectric (including quartz crystal microbalance biosensors) based on their transduction or as immunosensors, aptasensors, genosensors and enzymatic biosensors based on their recognition elements, respectively when are used antibodies, aptamers, nucleic acids, and enzymes. The majority of biosensors in environmental monitoring are recognized as immunosensors and enzymatic biosensors, but the development of aptasensors has recently increased due to the beneficial characteristics of aptamers, like ease of modification, thermal stability, in vitro synthesis and the ability to design their structure, to differentiate targets with different functional groups and to rehybridize.

Study on the design of biosensors for the monitoring of organic pollutants, potentially toxic elements and pathogens in the environment has led to the sustainable development of civilization due to the environmental pollution issues confronting human health. Various chromatographic techniques (such as gas chromatography and high-performance liquid chromatography combined with capillary electrophoresis or mass spectrometry) are conventional analytical methods used for environmental monitoring of pollutants, but they require costly reagents, time-consuming sample pre-treatment and costly equipment. Therefore, for monitoring pollutants responsible for adverse effects on habitats and human health, more sensitive, cost-effective, fast, easy to function, and compact biosensing devices are desperately needed to overcome the magnification of environmental issues. In the case of accidental release of pesticides or acute poisoning, for example, common methods are not appropriate for in situ measurements where fast, miniaturized, and portable equipment like environmental monitoring biosensors is required. In this regard, the role of nanotechnology in the creation of rapid and intelligent biosensing devices is crucial for the success of environmental pollutant detection; most recent biosensors include nanomaterials and novel nanocomposites in their systems, which are beneficial for improving analytical performance, such as sensitivity and detection limits.

For the detection and monitoring of different environmental pollutants, biosensors, including immunosensors, aptasensors, genosensors and enzymatic biosensors have been documented using antibodies, aptamers, nucleic acids, and enzymes as recognition elements.

Pesticides

Pesticides are among the most significant environmental pollutants because of their large presence in the environment. Organophosphorus insecticides, for example, are commonly used in agriculture and represent a group of pesticides which, due to their high toxicity, are of immense environmental concern. Easy, responsive, and miniaturized in situ methodologies like biosensors have therefore been established as analytical strategies for their detection and monitoring, without the need for comprehensive sample pre-treatment.

Disposable amperometric enzymatic (acetylcholinesterase) biosensors were proposed for the detection of organophosphorus insecticides using paraoxon as a model analyte applying a cysteamine self-assembled monolayer on gold screen-printed electrodes. The disposable biosensors showed a linear spectrum of up to 40 ppb with a 2 ppb detection limit and a 113 μA mM cm^-2 sensitivity. Using the self-assembled monolayer, good analytical output could be due to the highly oriented enzyme immobilization. Recoveries of 97 ± 5 percent (n = 3) were reported after being tested in river water samples spiked with 10 ppb of paraoxon, indicating the effectiveness of such enzymatic biosensors. Furthermore, the use of disposable screen-printed electrodes dispenses with time-consuming methods like the reactivation of immobilized enzymes utilizing, for example, obidoxime solution and pralidoxime iodide (PAM) or the use of the renewable enzyme membrane needed for the second application of biosensors.

Nanoparticles based on iridium oxide have been used in the disposable enzymatic biosensor with tyrosinase based on low-cost screen-printed carbon electrodes for the detection of chlorpyrifos in river water samples. Linear biosensor response (0.01–0.1 μM) and low detection limit (3 nM) were reported, which could be due to the high conductivity of nanoparticles of iridium oxide and tyrosinase efficiency. Recovery tests were carried out in river water samples with the addition of 0.1 μM of chlorpyrifos and recoveries of 90 ± 9.6 percent were obtained with a residual standard deviation (RSD) smaller than 10 percent (n = 3) to demonstrate the applicability of the biosensor.

Acetamiprid was detected by colorimetric aptasensors and water samples by impedimetric aptasensors in real environmental samples, like fresh surface soil samples. A linear range of 75 nM to 7.5 μM and a detection limit of 5 nM were observed with the colorimetric aptasensor, while a wider linear range (50 fM to 10 μM) and a lower detection limit (17 fM) were observed with the impedimetric aptasensor. Gold nanoparticles, multi-walled carbon nanotubes (MWCNT) and reduced graphene oxide nanoribbons were used in that biosensor as a composite to sustain the electrode surface acetamiprid aptamer, which could be responsible for higher electron transfer and improved analytical performance of the biosensor. A related detection limit (33 fM) was observed by an aptasensor based on silver nanoparticles anchored on nitrogen-doped nanocomposite graphene oxide constructed for acetamiprid detection in wastewater samples.

Pathogens

The existence of pathogens in environmental matrices, and especially in water compartments, could pose a serious risk to human health, and some biosensors have recently been suggested for monitoring the environment. For example, for the detection of metabolically active Legionella pneumophila in complex environmental water samples, rapid and precise optical biosensors based on surface plasmon resonance have been proposed. In one study, the detection principle was based on the identification of bacterial RNA by the immobilized RNA detector probe on the gold surface of the biochip. For signal amplification, streptavidin-conjugated quantum dots were used, and the detection period was approximately three hours, indicating the viability of the biosensing device for successful bacteria detection in the range of 10⁴-10⁸ CFU mL^-1.

Potentially toxic elements

The contamination by heavy metals and corresponding ions of the natural waters can pose significant risks to human health, and compact, low-cost, and rapid heavy metal analyses are a global priority concern. As a model target for testing an optical DNA biosensor for the detection of heavy metal ions that are extremely toxic and common pollutants in the environment, mercury ions (Hg²⁺) were used. The biosensor was compact, low-cost, and rapid with in situ screening of Hg²⁺ in natural waters in less than 10 min. The detection principle is focused on the capacity of certain metal ions to bind selectively to certain bases to form stable metal-mediated DNA duplexes; in the case of Hg²⁺, thymine bases can be selectively coordinated to form stable thymine-Hg²⁺-thymine complexes. In the detection range between 0 and 1000 nM, a detection limit of 1.2 nM was achieved, which is lower than the maximum value requested by the United States Environmental Protection Agency (10 nM)

For the detection of Pb²⁺ in water samples (pond and lake water samples) using DNAzymes/carboxylated magnetic beads and DNA aptamers, two fluorescence based optical biosensors have recently been suggested. The detection limits of 5 nM and 61 nM, respectively, were observed by biosensors based on DNAzymes and DNA aptamers, with a respective linear detection range of 0 to 50 nM and 100 to 1000 nM. The use of label-free unique dye (SYBER Green I), which was intercalated with double stranded DNA, showing strong fluorescence intensities, as seen in Figure 5. Moreover, the absence of biosensor fluorescence intensity is observed only with the dye (curve a). With the DNAzyme + Pb²⁺ (curve b) the fluorescence intensity increases with the addition of the dye + DNAzyme + Pb²⁺, illustrating the sensitivity of the biosensor towards Pb²⁺.

Figure 5. Fluorescence emission spectra for detection of Pb²⁺

Toxins

Harmful toxins like brevetoxins and microcystins are created by the eutrophication of aquatic systems by the algal blooms of cyanobacteria, and thus accurate and cost-effective systems are needed for the early detection of such toxins. For the sensitive detection of brevetoxin-2, a marine neurotoxin, an electrochemical aptasensor has been used composed by gold electrodes functionalized with cysteamine self-assembled monolayers. A detection limit of 106 pg mL^-1 was achieved and strong selectivity was observed for brevetoxin-2 against other toxins of various groups, like okadaic acid and microcystin. The feasibility of the aptasensor for detecting brevetoxin-2 in real samples was achieved by analysing shellfish and strong recoveries (102-110 percent) were reported, indicating no interaction with the aptasensor response from the shellfish matrix.

Endocrine disrupting chemicals

In water samples, bisphenol A was detected as an endocrine disrupting chemical by aptasensors based on the fluorescence principle with functionalized aptamers (fluorescein amidite) and gold nanoparticles and based on evanescent-wave optical fibre. The evanescent-wave optical fibre aptasensor was compact and found to be rapid, cost-effective, sensitive and selective for the detection of bisphenol A in water samples, with the benefit of no requirement of any pre-concentration or treatment steps.  Furthermore, the aptasensor can be reused for 90 s by regeneration with a 0.5% sodium dodecyl sulphate (SDS) solution and further washing with a phosphate buffered saline (PBS) solution (pH 7.2) for over a hundred assay cycles without any noticeable loss of efficiency. Similar detection limits (0.1 and 0.45 ng mL^-1) were observed in both optical biosensors where the DNA molecule probe, which is the complementary sequence of a small fraction of the bisphenol A aptamer, was adsorbed by electrostatic interaction in the surface of gold nanoparticles and covalently immobilized on the surface of the fibre. Lately, for the detection of bisphenol A in river water samples using molybdenum carbide nanotubes, another fluorescence-based aptasensor was proposed. With such a label-free, inexpensive, and easy to use aptasensor, a low detection limit of 0.23 ng mL⁻¹ has been obtained. The specificity of the aptasensor was evaluated by analysing other molecules with structures similar to that of bisphenol A (e.g., 4,4J-biphenol, bisphenol AF, and 4,4J-sulfonyldiphenol) and only background signals showing high specificity for bisphenol A were identified for these molecules.

A disposable and label-free electrochemical immunosensor based on a field effect transistor with SWCNT has recently been employed in seawater samples for assessing another endocrine disrupting chemical – 4-nonylphenol. The immunosensor has a high reproducibility (0.56 ± 0.08%), an average recovery of 97.8% to 104.6% and a low detection limit (5 μg L^-1), which is lower than the recommended maximum concentration of 7 μg L^-1 specified by the corresponding regulations. In seawater samples such as 4-nonylphenol, the biosensor could be used to detect hazardous priority substances, even at low concentrations and with an easy and low-cost methodology.

Other environmental compounds

New, fast, and accurate analytical methodologies have been needed for the early detection and monitoring of various other hazardous compounds liberated during algal blooms. Due to the excellent sensitivity and specificity of nucleic acid probes to their complementary binding partners, biosensors have been developed to detect algal RNA. For the enhanced selective and sensitive detection of RNA from 13 harmful algal organisms, an electrochemical genosensor based on screen-printed gold electrode was recently reported; the genosensor could distinguish RNA targets from environmental samples (spiked seawater samples) containing 10⁵ cells, considered to be the limit of detection.