11 August 2016

Agricultural Technology, commonly referred to as ‘agri-tech’ is a new scientific discipline aimed to address the emerging threat of food shortage across the globe. It relies on novel genetic, chemical, robotic and engineering approaches in order to permit sustainable intensification of farming across crops, livestock and fisheries. This is in part due to a dramatic rise in world population and a surge in demand for the ‘western diet’ across the developing world.

The growing requirement for food has also meant that new methods are required for detection of food contaminants and toxins. These challenges place further pressure on the agricultural sector to  address the global challenge of food security. Thus, the development of new and innovative technologies is critical to constantly monitor food safety measures and fulfil the growing agricultural demands in a sustainable fashion.

Challenges facing the Agricultural Industry

The world’s population is expected to significantly rise from 7.2 billion to between 9 and 10 billion people by 2050 (United Nations, 2013). As a result, the food market has become increasingly globalised, with food safety issues a critical component in meeting the consumer’s high expectations.

Food safety faces many challenges which will continue to arise in unpredictable ways, largely due to (CDC, 2016):

  1. Changes in food production and supply.
  2. Increased food imports.
  3. Changes in environment leading to food contamination.
  4. Finding more multi-state outbreaks.
  5. New and emerging bacteria, toxins, and antibiotic resistance.
  6. Unexpected sources of food-borne diseases.

To date, multiple varieties of agricultural pollutants such as pesticides residues, chemical raw materials, industrial dye, organic pollutants and toxins produced naturally in food (aflatoxins) have all been detected in foods, resulting in food safety fears (Liu and Zhang, 2015). Due to the numerous drawbacks and disadvantages of existing analytical methods utilised such as chromatography, mass spectroscopy, and enzyme linked immunosorbent assays (ELISA) for food safety detection, there is an urgent need to develop scientific and efficient methods to address the ever growing food safety concerns. Current challenges and drawbacks of existing food safety detection methods include:

  1. High cost analysis equipment

Much of the equipment required for current testing procedures relies on expensive, laboratory based machinery. This is an important issue for food procedures in less economically developed countries who lack the necessary finance to afford running these tests.

  1. Low sensitivity and specificity

Current established methods of detection lack the required sensitivity and specificity which may ultimately lead to relatively poor sensitivity for many targets. This may result in false positive/ negatives to be given which can cause major safety concerns in food safety.

  1. Time consuming detection

Traditional assays and analytical tool used for current testing procedure are tedious and time-consuming which further delays results and outcomes. This can be largely problematic for food detection due to the downstream effect of contamination of stock food yields.

  1. Complicated operations

The complication behind current testing methods include strict laboratory testing, portability, and difficulty of use which significantly restricts many farmers. These disadvantages can ultimately draw further complications due to the lack of efficiency and effectiveness of current analytical ad testing tools in food analysis.

  1. Difficulties in rapid monitoring

Rapid monitoring and efficiency is highly sought for agricultural and food safety testing to analyse and speed up more stock. The lack of rapid monitoring can ultimately influence safety and health outcomes of many food products which can have downstream detrimental effects. As a result, agri-tech requires new rapid and effective detection methods with high sensitivity, low detecting limits, uncomplicated operations and low cost.

Aptamers and Agri-Tech

The aptamer technology is a newly developed detection and analysis method in food safety, agricultural monitoring, and pesticides and nutrient control strategies (Liu and Zhang, 2015) and has already been used in a number of different applications. Importantly, aptamers are biodegradable and do not affect animal or aquatic organism health unlike other tests.

Aptamers are short strands of nucleic acids that serve to bind to a target with high affinity and specificity and as such, are commonly referred to as ‘synthetic antibodies’. Aptamer targets include proteins, cells, and even small molecules which are typically problematic to antibodies. In theory, almost any target molecule can be selected, with aptamers previously having demonstrated high recognition and binding capability to low molecular mass substances (Osborne and Ellington, 1997). As a result, aptamers serve as useful tools for the identification, separation and purification of molecules and have become popular in the biomedical space (Liu and Zhang, 2015). It is also possible to further enhance the stability and improve other molecular characteristics of aptamers by developing their resistance to ion intensity, temperature, and acidity-alkalinity (Liu and Zhang, 2015).

Aptamer based assays have already demonstrated increased applicability in diagnostics, environmental or food quality testing, with analytical performance rivalling those of antibody-based assays. Some of the limitations of many conventional assays can thus be circumvented by aptamers as alternative recognition reagents (Marimuthu et al., 2012). Although the application of aptamers to poisonous and harmful materials in food is now at initial stages, existing studies and findings supplement theoretical basis and technical support for the actual application of aptamer technology in various agri-tech fields and food detection as highlighted in figure 1.

Food Contamination

Figure 1 Aptamer in food safety and sensor development

Microorganisms in Food

Traditional testing methods for food microbial rely on ‘plate counting’ which has the main disadvantage of a long detection cycle and a complex readout (Nugen & Baeumner, 2008). Unlike plate counting, aptamers can yield information quickly and simply and has been successfully used to detect multiple pathogens. Salmonella, an important foodborne pathogen that pose a serious threat to food safety has been studied whereby Joshi  et al. (2009) have previously selected and assessed DNA aptamers for the capture and detection of Salmonella enteric serovar Typhimurium. The study indicated the applicability of Salmonella-specific aptamers for pre-analytical sample processing as applied to complex sample matrices. Similarly, Hyeon et al. (2012) demonstrated the potential of RNA aptamers in detecting Salmonella enteritidis with high specificity and observed no cross reactivity to other Salmonella Servar strains.  Using a Salmonella specific recognition aptamer, Ma et al (2014) was able to construct an electrochemical biosensor for Salmonella detection with low detection limits reaching as low as 3 CFU/mL.

Other food pathogens have also been targeted for developing rapid detection methods. Bruno et al (2009) developed a quntum dot (QD)-based sandwich assay for Campylobacter jejuni detection (a common contributor to gastroenteritis) with detection limits as low as 10-25 CFU in various food matrices. Similarly, Wu et al (2010) developed an aptamer nanoscale polydiacetylene based biosensor for rapid detection of E. coli at concentrations of 105 CFU/ml. It is therefore evident that aptamer based detection of food pathogens has significant potential to help alleviate food health concerns through a simple and rapid technology.

Heavy Metal Ions

Industrial derived emissions like cadmium, mercury, lead, and other heavy metal ions entered into river and soil can cause major pollution concerns. This could accumulate in crops, animal feedstock and other raw material entering the human food chain which may potentially cause acute or chronic poisoning in humans (Lin et al., 2011). The main source of high heavy metal ion concentrations in food stem from pesticide use , industrial and raw material pollution as well as additives and packaging material in food processing (Liu and Zhang, 2015). Thus a rapid testing technology for heavy metal ions is urgently required for food samples as few applications in food analysis are reported. Ye et al (2012) developed a robust method for the visual detection of heavy metals ions such as mercury (Hg2+) and lead (Pb2+) using aptamer-functionalised colloidal photonic crystal hydrogel (CPCH) films. The CPCHs were derived from a colloidal crystal array of monodisperse silica nanoparticles, which were polymerized within the polyacrylamide hydrogel and cross linked in the hydrogel network with with heavy metal ion responsive aptamers as shown in Figure 2.

Agr-Tech 4

Figure 2 Visual detection of heavy metal ions using aptamer-functionalized colloidal photonic crystal hydrogel (CPCH) films (Ye et al (2012).

This CPCH aptasensor demonstrated the ability to screen a wide concentration range of heavy metal ions with high selectivity and reversibility in food and the environmental sample screening.

Bio-toxins

Bio-toxins in microbial metabolic products have very strong toxicity, mostly with carcinogenic, teratogenic, and mutagenic effects (Chu, 1991). Aptamer specific for targets such as ricin, cholera toxin, staphylococcal enterotoxins, ochratoxin A (OTA), and abrin toxin have been raised and selected for different detection methods (Tombelli, Minunni,and Mascanni, 2007). Haes et al. (2006) demonstrated the ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers which offers a promising method for selective, rapid, and sensitive detection of ricin for food safety analysis.

OTA, a mycotoxin produced by several species of Aspergillus and Penicillium can be found in many foods including cereals, cocoa, coffee, spices and dried fruits (Liu and Zhang, 2015). OTA has proven nephrotoxic and carcinogenic and poses a serious health to humans. OTA has been detected in various agro-products and foods, and feed sources and so far, numerous aptamer based assays have been developed for OTA detection (Liu and Zhang, 2015). OTA aptamers have been selected with high specificity and have been used for the determination of parts per billion (ppb) quantities of OTA in naturally contaminated wheat samples (Cruz-Aguado and Penner, 2008). Similarly Kuang et al (2010) developed a ultrasensitive and rapid electrochemical sensor for the specific detection of OTA with sensitivity as low as 30pg/ml.

Aflatoxins on the other hand are secondary products metabolised by Asperigillus flavus and Apergillus parasiticus which a classed as highly toxic and carcinogenic (Liu and Zhang, 2015). Using immobilised aptamers as affinity capture reagents, an electrochemical Fe3O4 / Polyanaline-based aptamer sesnors (Aptasensor) for label free and direct detection of aflatoxin M1 (AFM1) was developed which revealed a  detection limit of 1.98 ng/L in the range of 6-60ng/L (Nguyen et al. 2013).

Other food biotoxins including Botulinum toxin, Zeralenone, and staphylococcus aureus have been also targeted by aptamers. Aptasensors have also been developed against these targets to provide significant improvements in quality control and food safety through simple rapid and sensitive testing system for agricultural product monitoring (Liu and Zhang, 2015).

Antibiotics

Food quality and safety issues caused by excessive antibiotic residues have recently aroused significant attention. A large and compelling body of scientific evidence demonstrates that antibiotic use in agriculture contributes to the emergence of resistant bacteria and their spread to humans (Liu and Zhang, 2015). Currently, methods used for the detection of antibiotics include liquid chromatography, mass spectroscopy and enzyme linked immunosorbent assays, although new evidence suggests that aptamers may be able to replace many of these more complex tests. For example, one recent study used RNA aptamers to detect levels of the veterinary antibiotic danofloxacin present in food products showing both high affinity and specificity (Han et al. 2014). Moreover, aptamers have also been identified for antibiotics for human use – tetracycline, oxytetracycline and doxycycline and have been touted as targeted delivery tools, along with food contamination sensors (Wang et al., 2014).

Pesticides and Fertilisers

The overreliance on pesticides for intensive farming purposes such as carbamate has become an increased concern (Wang et al., 2011). At present, the main detection methods of pesticide residue are chromatography, spectral analysis, enzyme inhibition assays and immune analysis which have many drawbacks (Wang et al., 2011). Numerous studies have successfully isolated aptamers for specific pesticides including acetamiprid with a Kd 4.9μm (He et al., 2011) and DNA aptamers to discriminate against four organophosphorus pesticides including phorate, profenofos, isocarbophos, and omethoate (Pang  et al. 2014). Moreover, this method was validated in contaminated apple juice which further demonstrates the wide spectrum applicability of aptamers (Liu and Zhang, 2015).

Likewise, the excessive use of fertilizer has posed considerable environmental threats. The amount of nitrogen applied to crops now far exceeds the nitrogen utilized, leading to excess nitrates, gaseous ammonia, and nitrogen oxides present within the environment (DeRossa et al., 2010). Aptamer nanotechnology has therefore been utilised to synchronise the release of nutrients with the plants uptake of nutrients. This novel technology implies the use polyelectrolyte microcapsules containing aptamers in their walls that are specific for key plant signals (DeRossa et al., 2010). This allows the delivery of nutrient molecules within the microcapsules only once required by the plants. Root exudate specific aptamers thus act as molecular recognition probes in the development of an intelligent fertiliser system which would help alleviate the growing concerns behind the excessive use of fertilisers and their long-term damage to the ecosystem and environment.

Allergens

Abnormal immune mediated reactions from food allergies which occur in individual after consumption of certain food or food ingredients including wheat, peanuts, soy that can life threatening for some individuals (Turner et al., 2016 ). As most allergens are proteins, aptamers have been selected to bind to allergic proteins such as peanut allergen Ara h 1 within nano-molar range while discriminating Araa h 1 to Ara h 2 (Trans et al., 2013). One major hurdle of effective management of celiac disease is the sensitivity of the available methods for assessing gluten contents in food. Amaya-Gonzalez et al (2014) successfully selected aptamers to celiac disease-triggering hydrophobic protein gliadin. By using aptamers as specific receptors, a highly sensitive approach for gluten analysis was developed which allowed measurements of as low as 0.5 ppb of gliadin standard (0.5 ppm of gluten) which is 6 fold higher sensitivity than reference immunoassay (Amaya-Gonzalez et al., 2014).

Conclusions

Accompanied by industrial globalization, rapid urbanization, and population increment, mass production and staple trading for food consumption are up soaring continuously. Foodborne disease resultant from various food safety issues is currently a crucial public health concern worldwide, which has not only created a great burden on both economy and society, but also greatly threatens the sustainability of the agricultural industry. Nowadays, as complementary measures to and with advantageous merits over classic analytical methods, highly specific and selective aptamers have found their increasingly important roles in various domains of Agri-Tech and food analysis. This generates much optimism to resolve the heightened problems and challenges faced within the agricultural and food production industries.

Aptamer group

Aptamer Group takes a high-throughput approach using liquid handling robotics and dedicated researchers to identify aptamers against novel and significant targets. We are committed to finding the perfect aptamers to your target and use a proprietary selection technique to identify high affinity aptamers with specificity in as short as 3 months.

The aptamer group is committed to the development of test kits, and test procedures that are simple, robust, safe, and reliable enough to be performed routinely on site to improve testing and quality control throughout agricultural value chains. Aptamer Group’s biomarker discovery, diagnostic and therapeutic divisions aim to conduct further research by focusing on agricultural opportunities in combination with innovative approaches to help overcome the numerous challenges faced in both the Agri-Tech and food safety sector.

For further enquiries, please contact us on info@aptamergroup.co.uk

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