Reproducibility of sequential ssDNA detection in human serum
Products & Applications
Biosensing techniques such as Surface Plasmon Resonance imaging (SPRi) are widely used for monitoring biological interactions in real time. For reliable results, the biochip surface must promote specific interactions between the immobilised ligand and the analyte in solution while minimising off-target binding events.
The goal of this study was to demonstrate the robustness of K-One, Kimialys’ proprietary surface chemistry, applied to gold biochips for SPRi analysis. The study was conducted by repeating multiple cycles of DNA hybridisation and surface regeneration on the same K-One-pre-functionalised SPRi biochip, using human serum as the DNA diluent. The resultant data (collected using a Horiba XelPleX for multiplexed analysis) showed no loss of specific signal or increase in background noise over the course of the experiment, even at elevated serum concentrations.
Proof of Concept
To establish experimental conditions, 1 µM of a 50-base single-stranded DNA (ssDNA) was immobilised on defined regions (spots) of the SPRi biochip surface. Spots comprising immobilised non-complementary ssDNA served as a control, while background signal was measured directly on the biochip surface. Following injection of 50 nM complementary ssDNA (in human serum diluted 100x), binding data were recorded in the form of a sensorgram (Figure 1).
Figure 1. Sensorgram data for a single cycle of DNA hybridisation and regeneration. 1) sample injection, 2) association, 3) sample injection is stopped, and the biochip is rinsed with running buffer, 4) dissociation, 5) regeneration, 6) biochip is rinsed with running buffer.
Injection of the sample resulted in a rapid increase of the overall signal intensity due to the increased refractive index (1). The background and non-complementary ssDNA signals then reached a plateau, indicative of the absence of interaction at these selected areas, while the signal detected on the complementary ssDNA spots continued to rise due to specific hybridisation of the two complementary ssDNA strands (2). During the dissociation phase, the background and non-complementary ssDNA signals decreased rapidly to the baseline level, whereas the signal intensity on the complementary ssDNA spot remained at a constant high level, confirming the formation of dsDNA (4).
Consecutive cycles of hybridisation and regeneration in human serum (diluted 100x)
The robustness of the K-One surface chemistry was evaluated by performing 12 consecutive cycles of DNA hybridisation and regeneration, using 50 nM ssDNA in human serum diluted 100x and measuring the signal at 500 s, during the dissociation (Figure 2).
Figure 2. 12 consecutive cycles of DNA hybridisation and regeneration in human serum diluted 100x. Dotted lines correspond to the linear regression.
The data shows that the biochip retains its capacity to capture the target ssDNA, with no increase in non-complementary ssDNA binding over multiple cycles of hybridisation and regeneration. This demonstrates the robustness of the K-One surface chemistry and assures a constant measurement sensitivity.
Extended biochip use with complex biological samples (human serum diluted 20x)
To further assess the durability of the K-One surface chemistry, 39 consecutive cycles of hybridisation and regeneration were performed in human serum diluted 20x (Figure 3).
Figure 3. Thirty nine consecutive cycles of DNA hybridisation and regeneration in human serum diluted 20x.
The extended number of cycles and increased serum concentration had no impact on biochip performance, proving experimental sensitivity had been maintained despite experimental conditions being more challenging.
This study demonstrates the robustness of K-One surface chemistry under repeated cycles of DNA hybridisation and regeneration using SPRi technology. Importantly, K-One surface chemistry is suitable for a large variety of ligands (oligonucleotides, proteins, antibodies, peptides, enzymes) and, as shown here, provides exceptional specificity and reproducibility in complex biological samples. These benefits allow for unprecedented SPR analysis, paving the way for further use of this well-known technique for innovative biosensing and diagnostic applications.