The project involves three major components:
In this project, we completed the biosensor development, integrated it with an app for color-based analysis using smartphone photos, and successfully designed and tested the plasmid for E. coli transformation. These achievements set the stage for further refinement of the system in the future.
Anabaena Sensory Rhodopsin (ASR) is a photoreceptor that can regulate gene expression through its C-terminal domain. Proteins typically have two ends: the N-terminal (head) and the C-terminal (tail). In the case of ASR, the C-terminal can bind to DNA, specifically to the cpcB promoter in this system, to inhibit gene expression.
ASR is specifically sensible to light with a wavelength around 550 nanometers, where the difference in gene expression is most pronounced. Other wavelengths of light are less effective for controlling ASR probably due to the evolutionary advantages, because they are more likely to be absorbed by photosynthetic pigments, reducing ASR's ability to regulate gene expression.
When exposed to the light, the retinal molecule within ASR undergoes a transformation from its all-trans form to its 13-cis form. This change causes the C-terminal of ASR to detach from the DNA, allowing the gene to be expressed normally. A reporter gene is often included to indicate whether the protein has been successfully activated.
On the other hand, in the absence of light, ASR suppresses the expression of the reporter gene, keeping the system inactive. However, upon exposure to the light, ASR allows the gene to be expressed, meaning the cpcB promoter is activated, and gene expression can proceed.
Scheme 1. Biosensor design.
The biosensor system employs a probe immobilized on streptavidin-coated magnetic beads (SA-MBs), initially bound to an initiator sequence. Upon target recognition, whether a miRNA or protein, the initiator is displaced and released into solution. Magnetic separation is then performed to isolate the free initiator (I), which subsequently triggers a hybridization chain reaction (HCR). The HCR products incorporate split G-quadruplex structures that, in the presence of hemin, act as DNAzymes to catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H₂O₂). This catalytic process results in a colorimetric change, where the initially transparent solution turns blue. To stop the reaction, 0.2 M sulfuric acid is introduced, converting the solution to yellow. The intensity of the yellow color, measured at 450 nm, is directly proportional to the concentration of the target analyte, providing a quantitative readout of the assay.
Magnetic beads (MBs) play a crucial role in our biosensor design, enabling the purification, concentration, and efficient separation of cells, organelles, and biomolecules such as nucleic acids and proteins. MBs, composed of iron oxide, offer a highly effective means of isolating target molecules. This significantly reduces background noise and enhances the signal-to-noise ratio, making detection more reliable, especially in samples with low-abundance targets. By enriching trace target molecules prior to analysis, the overall sensitivity of the biosensor is improved.
The versatility of magnetic beads allows them to be integrated with a wide variety of biomolecular probes, making them adaptable to multiple targets, including proteins and nucleic acids. In our system, we utilized streptavidin-coated magnetic beads (SA-MBs) for target recognition. Through the strong and specific biotin-streptavidin interaction, our biotinylated DNA probes are immobilized onto the beads, ensuring precise capture of target molecules. This method enhances the overall detection accuracy, providing a highly reliable and adaptable platform for diverse biosensing applications.
Our 3WJ binary probe system is designed to allow for rapid and efficient adaptation to different target miRNAs. By making minimal adjustments to the complementary sequence (the pink target in the figure), we can easily develop specific detection probes for various miRNAs. This approach ensures that only minor changes are required for each new target, while the core structure of the probe remains intact. The system operates by releasing the initiator (gray) upon target recognition, which then triggers the HCR. Probes A (yellow) and B (black) form a binary complex that facilitates accurate target recognition.
This system’s versatility extends beyond miRNA detection. When detecting protein targets, the DNA probes can be replaced with aptamers, which are oligonucleotide sequences capable of binding to specific proteins with high affinity and specificity. Similar to miRNA detection, upon binding to the target protein, the aptamer releases the initiator, allowing the HCR to proceed. This adaptability means that the same binary probe system can be used for protein detection with only a simple modification—replacing the target-specific sequence with an aptamer, while the core detection mechanism remains the same.
The flexibility and scalability of this system make it a powerful platform for universal detection. Its ability to easily switch between miRNA and protein targets—whether for diagnostic purposes or research—demonstrates its potential as a versatile and efficient tool in biosensing technologies. The capability to detect both nucleic acids and proteins enhances its applicability in various biological and clinical settings.
We incorporate an enzyme-free, isothermal amplification nucleic acid strategy in our biosensor design. By relying on nucleic acid hybridization, it allows for efficient amplification with careful sequence design, providing a straightforward setup that avoids the complexities of enzymatic reactions. HCR offers a simpler and cost-effective alternative to Polymerase Chain Reaction (PCR) by operating at a constant temperature, eliminating the need for thermal cycling and reducing equipment complexity. The reaction is driven by enthalpy and entropy, meaning the stability of the DNA duplexes and the overall reaction dynamics are influenced by temperature. In HCR, the design of DNA sequences is crucial, as the reaction's efficiency depends on maintaining appropriate temperatures that match the designed conditions; hence, careful design and temperature control are essential to ensure the desired amplification and avoid issues related to sequence complementarity。
The use of G-quadruplex/hemin DNAzyme catalysis for the oxidation of TMB offers several advantages that make it highly suitable for rapid and straightforward detection systems. The most notable advantage is its capacity for naked-eye detection, where a visible color change—from transparent to blue and eventually to yellow—indicates the presence and concentration of the target analyte. This immediate visual assessment provides a simple, cost-effective solution without the need for complex instrumentation.
In addition to its visual detection capabilities, the G-quadruplex/hemin DNAzyme system can also generate electrochemical signals during the oxidation of TMB. This allows for more sensitive and precise measurements, with the potential for direct integration into an app-based platform. Similar to commercial glucose meters, this setup would enable real-time, portable diagnostics, where the signals are immediately quantified and analyzed on a smartphone, making it highly convenient for point-of-care applications.
A crucial aspect of our platform is its flexibility in target selection, allowing for the design of probes tailored to a wide range of biomarkers, including miRNAs and proteins. This versatility addresses a key limitation in single-target detection, where reliance on one biomarker could lead to false positives or insufficient specificity. By incorporating multiple biomarkers, we reduce the likelihood of false positives and improve the overall accuracy of detection. Additionally, our platform offers the advantage of testing clinically relevant miRNAs and proteins, providing a broader diagnostic application.
This capability is particularly advantageous for validating biomarkers identified in the literature. By comparing the detection of miRNAs or proteins in clinical samples, we can verify whether the proposed biomarkers truly correlate with the target condition. This not only strengthens the diagnostic power of our system but also ensures that the chosen biomarkers are reliable for real-world applications. In this way, our system serves as a robust, adaptable platform for multiplexed detection, offering enhanced precision in both research and clinical diagnostics.
To ensure the highest sensitivity and specificity in our biosensor, we carefully optimized several key reaction conditions.
In our design, we utilized a split G-quadruplex to reduce background noise and enhance signal specificity. By adjusting the split ratio of the G-quadruplex, we ensured that only when the target linker appears does the G-quadruplex form a stable complex with hemin and generate a strong signal in addition to TMB, and H₂O₂. This approach effectively keeps the signal low before target recognition. Furthermore, using the split G4 design minimizes the presence of G-quadruplex sequences in the HCR hairpins, preventing any potential interference with the efficiency of the HCR reaction.
In our experiments, we primarily focused on optimizing the reaction buffer composition to ensure the stability and efficiency of the HCR system. By fine-tuning the buffer concentration, we were able to achieve a high signal-to-noise (S/N) ratio, which is crucial for reliable detection. Through our tests, we confirmed that an optimal reaction time of just 30 minutes was sufficient to generate a robust S/N ratio, allowing for rapid detection without compromising accuracy.
To enhance the sensitivity and performance of our G-quadruplex/hemin DNAzyme system, we optimized several parameters involved in the TMB oxidation reaction. First, we adjusted the concentration of hemin, which plays a key role in catalyzing the oxidation of TMB, ensuring that the reaction proceeded efficiently without causing saturation or excessive background noise. Additionally, we optimized the amount of TMB substrate added, balancing the reaction kinetics to produce a clear and measurable signal while preventing overreaction.
We also fine-tuned the reaction time to allow sufficient oxidation of TMB, while ensuring the reaction didn’t run too long, which could lead to diminished specificity or increased background signal. Adjustments to the reaction buffer were made to support the catalytic activity of the G-quadruplex/hemin complex and maintain the stability of the reaction environment.
Furthermore, we evaluated the use of sulfuric acid as a stop solution to halt the reaction and improve the color contrast between samples. This step was particularly useful for achieving clearer differentiation in signal intensity, contributing to both qualitative and quantitative accuracy.
Our colorimetric assay design offers distinct benefits, particularly its capacity for naked-eye detection. This feature allows for rapid, straightforward analysis without the need for specialized equipment, making it accessible in various settings. The visible color change, from transparent to blue and then to yellow upon the addition of sulfuric acid, provides an immediate, qualitative indication of target presence. The color intensity correlates with target concentration, and the final yellow hue can be easily quantified through image capture using a smartphone camera. This enables the use of an app-based RGB analysis, further enhancing the precision of concentration measurements by translating color intensity into quantitative data, offering a more precise quantitative measurement. This dual approach of naked-eye detection and digital analysis makes the assay highly adaptable for point-of-care diagnostics, where quick, user-friendly results are essential. The use of G-quadruplexes enhances the assay's catalytic efficiency, and the visible color change can be easily captured and analyzed using basic tools, making this method both scalable and accessible.
In this experiment, we developed a G-quadruplex-based DNAzyme colorimetric biosensor for detecting cardiovascular disease biomarkers. The system uses magnetic particles for separation and an isothermal hybridization chain reaction (HCR) for signal amplification. It incorporates aptamer-based probes for protein detection and a three-way junction (3WJ) for miRNA detection, making it adaptable to different biomarker targets. .
Our biosensor integrates with an app, using smartphone cameras to analyze color changes from TMB oxidation for real-time, user-friendly detection. Performance tests confirmed its sensitivity for both naked-eye and app-based analysis. Future plans include integrating electrochemical detection, expanding its applications beyond cardiovascular disease. Overall, our biosensor offers a versatile, accessible platform for early disease detection.
