Breakthrough: Ultra-High Sensitivity Detection of Legionella Using an Aptamer-Based Electrochemical Sensor-Yeast Display Technology Special Topic-Tekbiotech-Yeast and Phage Display CRO, Expert in Nano-body and Antibody Drug Development

Breakthrough: Ultra-High Sensitivity Detection of Legionella Using an Aptamer-Based Electrochemical Sensor

I. Preface

Legionella pneumophila is a Gram-negative pathogenic bacterium that causes respiratory infections and, in severe cases, pneumonia and multi-organ failure. Consequently, researchers are continuously developing methods for the rapid and accurate detection of L. pneumophila in various settings. Various traditional detection methods exist, but each has associated limitations. For the first time, Aysha Shaukat et al. employed Cell-SELEX technology to screen for and identify AY-19, a highly specific aptamer. Based on this aptamer, they developed an electrochemical platform for the label-free, sensitive, specific, and rapid detection of L. pneumophila SG-1.

II. Research Highlights: The Powerful Combination of Aptamer and Electrochemical Sensing

1. Aptamer Identification – Cell-SELEX Screening

As shown in Figure 1(a), during each round of selection, the ssDNA library was co-incubated with L. pneumophila SG-1. After washing to remove unbound ssDNA, the bound DNA was recovered. Subsequently, the bound DNA was amplified by PCR and purified to form the new starting library for the next selection cycle. Figure 1(b) shows that the fluorescence intensity of bound ssDNA increased continuously over the first five rounds. In the fifth round, counter-selection was introduced using subspecies closely related to SG-1 to eliminate non-specific binding and enhance aptamer specificity. Finally, the ssDNA eluted from the tenth round was sequenced.

Figure 1: Schematic of the Cell-SELEX process and the fluorescence intensity of eluted ssDNA in each cycle

The authors further analyzed the sequencing results by aligning the selected ssDNA sequences using the multi-sequence alignment software PRALINE. As shown in Figure 2, conserved regions within each sequence are highlighted in red for clarity, while arrows indicate the nine sequences selected for further binding affinity studies.

Figure 2: Conserved sequences bound to SG-1

2. Aptamer Characterization

The authors used a fluorescence labeling method, labeling the 5' end of these nine selected sequences with FITC. These aptamers at specific concentrations were then incubated with a fixed number of SG-1 cells. After washing away unbound aptamers, the quantity of bound aptamer was measured by fluorescence intensity. Figure 3 shows the results for six aptamers, interpreted according to the equilibrium dissociation constant (Kd), which represents the aptamer concentration required to achieve half-maximal binding. The Kd values ranged from 14-75 nM, with AY-19 showing the lowest value (14.19 nM), indicating its strongest binding affinity.

Figure 3: Quantification of aptamer binding affinity by Kd value using fluorescence labeling

3. Sensor Construction

AY-19 was selected for fabricating the electrochemical aptasensor. As shown in Figure 4, AY-19 was thiol-modified by adding an -SH group at one end and drop-cast onto the surface of a gold electrode. After overnight incubation at room temperature, the -SH group formed an Au-S bond with the gold (Au), thereby immobilizing AY-19 upright onto the electrode surface. After aptamer immobilization, exposed gold regions remained on the electrode surface, which could non-specifically adsorb other substances, potentially causing false positive signals. Therefore, a small molecule, 6-mercaptohexanol (MCH), was used to "block" these vacant sites.

Figure 4: Schematic diagram of the fabrication of the electrochemical aptasensor

To verify the success of the fabrication steps, the authors used a redox probe molecule, [Fe(CN)?]3-/4-, to "interrogate" the electrode surface condition. The more easily electrons could pass through the electrode surface, the higher the current. Conversely, more surface modifications and greater negative charge hindered electron transfer, reducing current and increasing impedance. As the surface modifications increased, the measured current decreased (Figure 5a) and the impedance increased (Figure 5b), confirming that the aptamer and MCH were successfully immobilized on the electrode surface.

Figure 5: Characterization of the sensor construction process

4. Electrochemical Detection

To validate the sensitivity of the sensor in practical applications, the binding affinity was tested by varying the concentration of SG-1. As shown in Figure 6(a), increasing the SG-1 concentration led to a decrease in the current measured at the metal surface; the more bacteria bound, the more pronounced the decrease. The right panel plots the relationship between the change in current and the logarithm of the bacterial concentration, yielding a linear curve with a detection limit of 4.6 CFU/mL, demonstrating extremely high sensitivity.

Figure 6: Detection performance of the electrochemical sensor

To further validate the specificity of the electrochemical aptasensor, cross-reactivity experiments were performed. Various Legionella species were added alongside SG-1. As shown in Figure 7, the presence of other bacterial species had no significant effect on the results compared to the assay containing only SG-1, confirming the sensor's potential for accurate and reliable pathogen detection.

Figure 7: Specificity detection of the sensor using several *Legionella* species

This study obtained nine candidate sequences via Cell-SELEX technology, among which AY-19 exhibited the highest affinity, with a Kd value as low as 14.19 nM. AY-19 forms a typical stem-loop structure, offering better stability and stronger specificity than traditional antibodies, with no cross-reactivity with closely related species. Consequently, AY-19 was used to fabricate an electrochemical sensor for detecting SG-1, achieving extremely high sensitivity with a minimum detection limit of 4.6 CFU/mL, far superior to most existing methods.

TekBiotech (Tianjin) Co., Ltd. has accumulated years of project experience and expertise in nucleic acid aptamer selection. TekBiotech has established a comprehensive nucleic acid aptamer selection platform capable of providing high-quality aptamer screening services (including both RNA and DNA aptamers) for targets including but not limited to proteins, peptides, amino acids, and small molecule compounds. Additionally, TekBiotech can provide subsequent aptamer modification and functional validation services, delivering robust technical support for the research projects and drug development of our partners.

References

[1] Shaukat, A., Chrouda, A., Sadaf, S. et al. Author Correction: Cell-SELEX for aptamer discovery and its utilization in constructing electrochemical biosensor for rapid and highly sensitive detection of Legionella pneumophila serogroup 1. Sci Rep 14, 15951 (2024).

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