Biosensors

Rolling Circle Amplification

Lambert iGEM used rolling circle amplification (RCA) to detect miRNAs. Our padlock probes bind to their specific microRNAs (miRNAs) and yield RCP-fluorophore-quencher (RFQ) complexes which produce decreased fluorescence signals from background fluorophores. A decrease in fluorescence is expected as miRNA concentrations increase and bind to our rolling circle products (RCP).

There is a negative logarithmic correlation between the complement concentrations and the relative fluorescence units (RFU) (see Fig. 1). Moreover, the data shown above closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 2) correlating complement concentration to RFU (see Modeling). Therefore, the overall data collected depicts an accurate relationship between the miRNA concentration and RFU, further validating that RCA coupled with linear probes are an effective and efficient means of quantifying miRNA concentrations.

Figure 1. Characterization curve depicting the relationship between miRNA concentrations and fluorescence in RFUs of the linear probes

Figure 2. RCA model predicting the trend between miRNA concentrations (picomoles) and fluorescence output (RFU)

Padlock Probes

Lambert iGEM created Probebuilder as an alternative for generating padlock probes as compared to creating them by hand. We generated the padlock probe sequence for miR-451a on SnapGene (see Fig. 3). Next, we used the corresponding target miRNA and reporter sequences as inputs in Probebuilder to yield a sequence output (see Fig. 4). We compared the sequences created on both platforms and found that Probebuilder successfully developed a padlock probe identical to one created on SnapGene but in far less time (see Fig. 5). Additionally, we experimentally validated the padlock probe sensor, through rolling circle amplification and linear DNA probes.

Figure 3. Padlock probe sequence for miR-451a generated by hand on SnapGene

Figure 4. Automatically-generated padlock probe sequence for miR-451a after entering the input target miRNA sequence and reporter sequence on Probebuilder

Figure 5. Probebuilder-generated padlock probe sequences for miR-451a, identical to those produced by SnapGene

There is a significant decrease in the fluorescence intensity of a triplicate with FAM Probe, BHQ Probe, and the RCP produced as compared to a triplicate of just FAM tagged Probes (see Fig. 6). This finding experimentally validates the use of Probebuilder as a means of producing effective padlock probes.

Figure 6. Fluorescent reading of RCP produced by miR-451a padlock probe (generated via Probebuilder). BHQ-1 and FAM probes compared to solely FAM probes.

Hardware Quantification

Lambert iGEM designed a frugal handheld fluorometer, Micro-Q, to quantify fluorescence from our biosensors and help identify the concentrations of microRNAs (miRNAs) present. To validate Micro-Q’s efficacy in quantifying relative fluorescence, we compared fluorescence units with those of a commercial plate reader by paralleling units in readout (see Fig. 7).

Figure 7. Comparison of fluorescence values between Micro-Q and a commercial plate reader based on varying fluorescein concentrations

Sample Testing

To verify the practicality of our biosensors in the real world, Lambert iGEM experimented with pooled human serum as our proof of concept (see Fig. 8). We acquired this pooled human serum from a collaborating university.

Figure 8. Pooled human serum stock that was stored at -20°C

Upon consulting with Dr. Charles Searles from the Emory University School of Medicine we developed an experimentation protocol to test the ability of our rolling circle amplification sensors to detect microRNAs (miRNAs) in blood serum. Our protocol involved adding miRNA-1 and RNase inhibitors to serum samples, and running rolling circle amplification (RCA) reactions on these “spiked” serum samples. RNase inhibitor was added to prevent native RNases from degrading our added miRNA. We spiked the serum by adding 2 uL of 10nM miR-1 to a total 20uL reaction (1 nM). RCA was performed on this spiked serum (further diluting our miRNA to 40 pM), and the results were ran through gel electrophoresis. The gel electrophoresis results in Figure 9 show clear bands close to the gel, indicating our biosensors were able to produce a long strand, likely the rolling circle product (RCP).

Figure 9. Agarose gel with RCP from RCA reactions and negative control reactions that had no added miRNA from serum treated with 10 nM miRNA

To experimentally validate that the long DNA strand that was produced is our desired RCP, we utilized linear DNA probes as a means of reporting. These probes utilize adjacent FAM Dye Tagged Probes and BHQ-1 quencher tagged probes that are complementary to our RCP. In the presence of the RCP, the probes bind to the RCP, and the fluorescence produced by the FAM Dye is quenched by the BHQ-1 quencher, producing the effect of having a comparatively decreased fluorescent intensity.

As shown by Figure 10, there is statistically significant decrease in the fluorescent output of a triplicate with FAM Probe, BHQ Probe, and RCP as compared to a triplicate of just FAM tagged Probes. This confirms that we did produce our desired RCP in the RCA reaction performed on our miRNA-1-3p spiked serum. This further validates that biosensors utilizing RCA coupled with FAM/BHQ-1 linear DNA probes is an effective sensing and reporting mechanism for miR-1-3p.

Figure 10. Linear DNA Probe Fluorescence from Serum Extracted miRNA-1-3p Rolling Circle Amplification. Results show significant decrease in fluorescence, indicating a successful Proof of Concept.

Safety

Lambert iGEM was unable to extract serum from and test our reactions in whole human blood due to limitations of being a Biosafety Level 1 Laboratory (BSL-1). Therefore, we obtained pooled serum from a collaborating university, which contained no bloodborne pathogens and/or bioengineered products. While testing with serum, we wore personal protective equipment (PPE) including nitrile gloves, splash goggles/safety glasses, and lab coats to prevent body contact with and contamination of samples. Finally, we safely discarded the liquid samples following the disposal protocol for biohazardous liquids below (Vanderbilt University Medical Center, 2022):

Disposal Protocol

1. Have PPE on at all times
2. Store all treated serum samples in sealable glassware
3. Autoclave treated serum samples
4. After cooling, slowly drain samples down the lab sink while having PPE on
5. Wash your hands thoroughly

References