Introduction

Smallholder farmers are responsible for producing a third of the global food supply 1, yet they endure a slew of challenges ranging from climate change to financial instability 2. In order to safeguard against crop losses and maximise harvests, these farmers tend to overapply fertiliser - this is ultimately wasteful and contributes to environmental degradation such as eutrophication and air pollution 3 4.

To enable targeted application of fertilisers, these farmers require a way of accurately assessing crop health and nutrient needs. However, existing methods have their limitations:

  • Soil quality testing generally requires specialised equipment that may be expensive for smallholder farmers. Furthermore, soil testing assesses environmental factors and not crop health directly - drawing inferences from the former can therefore give rise to inaccurate crop health assessment.
  • Sampling and analysing crop tissue, on the other hand, requires expertise and is often performed off-site in laboratories. Shipping samples around is not only expensive but also time-consuming, meaning a significant delay between onset of nutrient deficiency symptoms and intervention.
  • Visual inspection of crops is often left as the only way for smallholder farmers to assess crop health. Unfortunately, this has a number of issues. First, a degree of subjectivity is introduced, where two farmers with different levels of skill and experience may assess crop health differently. Second, the symptoms of different nutrient deficiencies tend to overlap with each other 5, making it difficult for farmers to tell exactly which nutrient(s) are lacking. In this regard, this method fundamentally relies on onset of symptoms, meaning early detection and intervention is not possible at all.

Therefore, our team aims to leverage synthetic biology techniques to provide a comprehensive yet accessible crop diagnostic for smallholders farmers, by exploiting crop miRNA levels.

Our Solution - CamiRa

We aim to develop CamiRa - Crop Assessment via microRNA Analysis. CamiRa is an affordable, easy-to-use, all-in-one miRNA extraction and analysis kit which detects nutrient deficiencies before crops show visible signs for early intervention (i.e. before crop yield is affected). Due to its ease of use, CamiRa can also be used to check the crops after nutrient supplementation to check if they have recovered, to prevent overapplication resulting in eutrophication. In our design, the following considerations have been taken to accommodate the limitations of farmers:

  • Minimal liquid handling - For ease of use, our detection device is mostly paper-based
  • Safe chemicals - current established miRNA extraction techniques involve toxic and environmental-unfriendly chemicals (e.g. TRIzol)
  • No requirement for sophisticated lab equipment - 3D-printed centrifuge and Syringe pipette
  • Long shelf-life - lyophilised paper discs require less frequent purchases from farmers

Extraction

Our extraction protocol merges an isopropanol-based chemical precipitation with a hand centrifuge, designed to make a cheap, easy-to-operate and safe extraction procedure that could be carried out by farmers, without needing extensive laboratory equipment and training. None of the chemicals involved in the extraction process are hazardous, and can be easily disposed of.
This method relies on isopropanol’s ability to decrease the solubility of nucleic acids, allowing them to be pelleted out via a 15 min centrifugation step with the hand centrifuge. The pellet can then be resuspended in water for the detection test.

Detection

After extraction, the second part of our project is to develop a method of detecting miRNAs from our plant extracts. Three different detection methods have been considered: two using rolling circle amplification (RCA) as a form of isothermal signal amplification (G-quadruplex and Bioluminescence resonance energy transfer (BRET)), and one without amplification based on hybridisation.

Hybridization

Principle

We utilised sandwich hybridisation on Whatman filter paper discs to detect miRNA in plant extract, as shown in Figure 1. Half of the target miRNA sequence is hybridised to a single-stranded fluorophore-bound DNA probe. This leaves a single-stranded tail on the target miRNA which complementarily binds to a DNA probe immobilised on the paper discs with (3-Aminopropyl)triethoxysilane (APTMS) as a linker to the paper.

Figure 1: Sandwich hybridisation process, involving 1) APTMS addition to Whatman No.1 paper disc; 2) binding of DNA probe to paper disc; 3) binding of target miRNA to fluorophore-bound DNA probe; 4) binding of miRNA to immobilised DNA probe.

What users would need to do

Users will first dissolve the following 3 provided nucleic acids in deionised water:

  • Lyophilised fluorophore-bound DNA probe for the miRNA of interest
  • Fluorophore-bound miR16 DNA probe
  • hsa-miR16-5p standard

After executing the extraction protocol, users need to prepare a pre-hybridisation solution by combining the following, and incubate it for 10 min in the dark at room temperature:

  • Extraction lysate, fluorophore-bound DNA probe for the miRNA of interest, fluorophore-bound DNA probe for hsa-miR16-5p, hsa-miR16-5p standard, and the provided buffer solution

The test solution should be applied to each of 3 different paper discs and left to incubate for 10 minutes.

  • Test disc: paper disc containing an immobilised DNA probe for the plant miRNA of interest
  • Negative control disc: paper disc containing an immobilised DNA probe for another miRNA not present in the solution
  • Positive control disc: paper disc containing an immobilised hsa-miR16-5p DNA probe

Each disc should then be washed with provided wash solutions and left to dry in the dark for 1 hr.

Lastly, each disc should be inserted in a tray within a dark box with a built-in light source. The emitted light is subsequently quantified, and the test and control data are analysed, all using a smartphone application.

RCA/BRET, RCA/G-quadruplex

Principle

We employ a paper disc with components necessary for Rolling circle amplification (RCA), specifically polymerase enzyme (phi29 DNA polymerase) and circular DNA padlock probe that contains the complementary sequence to miRNA. As illustrated in Figure 2, RCA utilises miRNA as the primer 6 to replicate the DNA sequence on the circular probe, generating a long stretch of DNA. The extended single-stranded DNA is due to the strand displacement activity of phi29 DNA polymerase and is then co-opted for quantification of miRNA levels.

Figure 2: Rolling circle amplification (RCA) can be initiated by miRNA binding to a circular ssDNA probe, with extension subsequently performed by a polymerase.

For the first method, as shown in Figure 3, the stretch of DNA contains the repeated binding sites for zinc-finger proteins conjugated to either NanoLuc Luciferase (NLuc) (Donor) or mNeonGreen (mNG) fluorescent protein (Acceptor) in Bioluminescence resonance energy transfer (BRET). The donor or NLuc is supplied with its substrate and generates light as a result of chemical reactions (Luminescence). The acceptor or mNG is responsible for absorbing the luminescent light and emitting light at a longer wavelength.

Figure 3: Bioluminescence resonance energy transfer (BRET) occurring after co-localisation of NanoLuc Luciferase and mNeonGreen on an extended rolling circle amplification (RCA) product.

The second approach is to use a guanine-rich DNA sequence that forms a non-canonical secondary structure named G-quadruplex. A G-quadruplex consists of stacked G-quartets (four guanine bases) on the same ssDNA strand and is stabilised by the presence of monovalent cation e.g. potassium ion (K+) 7. This conformation of DNA can be detected using a fluorescent dye called Thioflavin T as seen in Figure 4. Not only is Thioflavin T water-soluble but Thioflavin T also exhibits low fluorescence background in unbound states and enhanced fluorescence intensity upon binding to G-quadruplexes 7.

Figure 4: Fluorescence resulting from excited fluorescent dyes bound to G-quadruplex structures formed from an extended rolling circle amplification (RCA) product.

What users would need to do

After executing the extraction protocol, users should add the plant extract to two different paper discs (“RCA discs”) impregnated with reaction components and circular probes. The two circular probes are designed for either miRNA representative of a particular nutrient stress or a spiked miRNA added during extraction to standardise the test for different extraction efficiencies. Two paper discs, one for each circular probe, are added with water as negative controls for each circular probe and serve to normalise the results for different conditions in different settings. The incubation step lasts for 2 hours prior to detection and can be done at room temperature.

The aforementioned system works for G-quadruplex assay, and its quantification entails the excitation of the dye in a dark box with a built-in light source. The emitted light was measured by a smartphone application.

Meanwhile, there is a second step to the BRET assay, involving another set of paper discs (“BRET discs”). BRET protein donor and acceptor, substrate of the donor and other components are freeze-dried onto these discs. After the previously described incubation step, users should proceed to transfer the BRET disc onto the RCA disc for a second incubation step of 30 minutes, also at room temperature. Afterwards, the longer wavelength emitted from BRET is detected and quantified via a smartphone application.

Gene ontology

As different specific miRNAs are upregulated with specific stress responses, candidate miRNAs with unique sequences ought to be chosen to reduce the likelihood of a false positive.

To this end, we developed a software tool for miRNA sequence and function comparison. With an organism selected, its miRNAs are all plotted on a graph based on their sequence similarity, before being clustered based on their annotation, obtained from gene ontology analysis. This allows for the identification of potential miRNA target candidates for diagnostic testing - miRNAs with the desired target response, but also low sequence similarity with unrelated miRNAs.

Outlook

We envision our kit as an accessible, point-of-care diagnostic tool for nutrient deficiencies in plants. Our kit would aid farmers with limited resources in preemptively identifying nutrient stresses before symptoms manifest in crops, increasing crop yields and reducing the inequalities that they experience due to lack of access to nutrient deficiency detection methods. Additionally, it also addresses overfertilisation, which leads to eutrophication and the destruction of aquatic ecosystems. Furthermore, more informed decisions on fertiliser applications reduce the cost and relieve the financial burden on farmers. Therefore, we believe in the potential of our product to create positive social, economical and environmental impacts.

Footnotes

  1. Ritchie, H. (2021, August 6). Smallholders produce one-third of the world’s food, less than half of what many headlines claim. Our World in Data. https://ourworldindata.org/smallholder-food-production ↩

  2. Touch, V., Tan, D. K. Y., Cook, B. R., Liu, D. L., Cross, R., Tran, T. A., Utomo, A., Yous, S., Grunbuhel, C., & Cowie, A. (2024). Smallholder farmers’ challenges and opportunities: Implications for agricultural production, environment and food security. Journal of Environmental Management, 370(122536), 122536. https://doi.org/10.1016/j.jenvman.2024.122536 ↩

  3. Sapkota, T. B., & Singh, B. (2025). India’s fertilizer policies: implications for food security, environmental sustainability, and climate change. Regional Environmental Change, 25(2). https://doi.org/10.1007/s10113-025-02395-9 ↩

  4. Ren, C., Jin, S., Wu, Y., Zhang, B., Kanter, D. R., Wu, B., Xi, X., Zhang, X., Chen, D., Xu, J., & Gu, B. (2021). Fertilizer overuse in Chinese smallholders due to lack of fixed inputs. Journal of Environmental Management, 293(293), 112913–112913. https://doi.org/10.1016/j.jenvman.2021.112913 ↩

  5. The Royal Horticultural Society. (2022). Nutrient deficiencies. https://www.rhs.org.uk/prevention-protection/nutrient-deficiencies ↩

  6. Li, Y., Zhou, L., Ni, W., Luo, Q., Zhu, C., & Wu, Y. (2019). Portable and Field-Ready Detection of Circulating MicroRNAs with Paper-Based Bioluminescent Sensing and Isothermal Amplification. Analytical Chemistry, 91(23), 14838–14841. https://doi.org/10.1021/acs.analchem.9b04422 ↩

  7. Renaud de la Faverie, A., Guédin, A., Bedrat, A., Yatsunyk, L. A., & Mergny, J. L. (2014). Thioflavin T as a fluorescence light-up probe for G4 formation. Nucleic acids research, 42(8), e65. https://doi.org/10.1093/nar/gku111 ↩ ↩2