Biochip surface chemistry robustness

Reproducibility of ssDNA detection on a biochip pre-functionalised with K-One surface chemistry, over several cycles of hybridization and denaturation in human serum

Introduction

A major challenge with surface-based biosensors is the reduction of non-specific interactions on the biochip surface whilst promoting specific interactions between the analyte in solution and the immobilized ligand. Kimialys proprietary K-One surface chemistry solves these problems.

This white paper aims to demonstrate the high reproducibility of K-One surface chemistry applied to gold biochips.

To determine the robustness of the K-One surface chemistry, a series of experiments were performed where cycles of DNA hybridization and denaturation were repeated on the same SPRi biochip. The ssDNA target molecule diluted in a human serum was flown across the biochip surface onto which the complementary ssDNA sequence had been immobilized using Kimialys surface chemistry. The amounts of non-specific and specific material retained at the surface were compared. Surface regeneration was achieved by exposure to high pH, known to denature double stranded DNA (dsDNA).

No loss of specific signal (DNA hybridization) or increase in non-specific background noise was observed over 40 cycles during the experiment.

Biosensor instrumentation: XelPlex (Surface Plasmon Resonance imaging) from Horiba

SPRi is an optical biosensing technique widely used to monitor biological interactions in real time. It requires a gold biochip surface functionalized with biomolecules or ligands that interact specifically with the target molecules present in solution. The detection of the dynamic of complex association and dissociation at the biochip surface leads to the characterization of the association and dissociation constants of the biological system, as well as the calculation of the affinity of the complex.

Thanks to the multiplexing capacity of SPRi technology, three separate measurements were analyzed in parallel:

  1. Specific interactions between the target ssDNA and the complementary ssDNA immobilized on the surface.
  2. Non-specific interactions between the target ssDNA and the non-complementary ssDNA immobilized on the surface.
  3. Background noise signal registered directly on the biochip surface where no ssDNA ligand is immobilized.

Experimental conditions

  • Ligand: 1 mM of a 50-base single-stranded DNA (ssDNA) was immobilized on the SPRi biochip surface via thiol-gold bound to the chip surface.
  • ssDNA spot definition:
Ssdna Spot Definition
Figure 1. Realtime image of the biochip surface after ssDNA deposition. Spots 6, 7, 11, 12, 19, 20 correspond to the complementary ssDNA of the target ssDNA. Spots 4, 5, 13, 14, 18, 21 correspond to the non-complementary ssDNA and spots 1, 2, 3, 8, 9, 10, 15, 16, 17 correspond to background.
  • Analytes:  50 nM of complementary ssDNA oligomer diluted in human serum (100x and 20x in PBS (Phosphate Buffer Saline)).
  • Running buffer: PBS, pH 7.4
  • Flow rate: 25 ul/min
  • Injection time: 200 seconds
  • Dissociation time: 800 seconds
  • Regeneration conditions: 50 mM NaOH
  1. DNA hybridization and denaturation in serum diluted 100x

Figure 2 shows a single experimental run; injection of the target ssDNA, dissociation phase and regeneration recorded as a function of time (SPRi sensorgram). The sensorgram corresponding to the hybridization of the two complementary ssDNA was compared with the sensorgram collected directly on the biochip surface (“background”). The interaction of the target ssDNA with the non-complementary ssDNA immobilized on the biochip surface was presented as a control.

First Cycle Of Target Ssdna Injection And Regeneration
Figure 2. First cycle of target ssDNA injection and regeneration. Sample: 50 nM ssDNA in human serum diluted 100x in PBS. Regeneration solution: 50 mM NaOH. Step 1 – fast increase of refractive index due to the injection of the sample; Step 2 – injection step: the sample solution flows through the biochip surface; Step 3 – injection of the sample is stopped. The biochip surface is then rinsed with running buffer; Step 4 – dissociation phase; Step 5 – regeneration phase; 6 – the surface is rinsed with the running buffer.
Biochip Surface
Figure 3. Images of the biochip surface during the injection of 50 nM ssDNA in human serum diluted 100x in PBS. 1 – Image difference before injection (Step 1); 2 – during the injection (Step 2); 3 – dissociation phase (Step 4) 4 – after regeneration (Step 6).

The first observation during the injection phase (step 1 in the Figure 2) is a rapid increase of the signal intensity due to the variation of the refractive index of the solution upon the injection of the human serum.

During the injection phase, the signal measured directly on the biochip surface (“background”) and on the surface where the “non-complementary DNA” was immobilized stabilized rapidly at t = 5 s until the end of the injection phase (t = 200 s).

Simultaneously, the signal intensity measured where the “complementary ssDNA” was immobilized continued to increase. The differential image 2 (Figure 3), corresponding to the image recorded during step 2 of the injection (Figure 2) confirms that all spots defined by the immobilized complementary ssDNA are clearly visible, whilst no spots are visible where non-complementary ssDNA was immobilized.

At t = 200 s the injection was stopped (Step 3) and the entire surface was rinsed with running buffer (PBS). The signal decreased rapidly due to refraction index variation between the serum and the PBS. The signal stabilized from approximately 250 s; and the intensity of the detected signal is dependent on the amount of material retained on the biochip surface (step 4, Figure 2 and image 3, Figure 3). 

The intensity of signal measured on the biochip surface or on the non-complementary DNA spots corresponded to the non-specific signal, whilst the signal detected on the complementary DNA spots corresponded to the specific signal as desired.

The injection of 50 mM NaOH (step 4) (just after t = 1000 s) allowed regeneration of the entire surface demonstrated by the return to baseline signalling levels immediately following injection (step 6, Figure 2 and image 4, figure 3).

Conclusion:

Despite the strong refractive index increase following injection of human serum, a clear contrast between the spots of the complementary ssDNA and the background chip surface was observed. During the dissociation phase, the complementary DNA spots remained clearly visible while the remainder of the chip surface returned to black, indicating that only the specific, desired signal was detected. The regeneration step was effective, demonstrated by the return of the entire chip surface to the initial state prior to commencement of the experiment.

2. Consecutive cycles of hybridization/denaturation in serum diluted 100x

Injection of 50 nM ssDNA diluted in human serum 100x was repeated on 12 consecutive occasions. The amplitude of the signal measured during the dissociation phase (500 s after the beginning of the injection of ssDNA) is plotted as a function of the consecutive injections (Figure 4).

Dna Hybridization
Figure 4. 12 consecutive cycles of DNA hybridization/regeneration. 50 nM of target ssDNA in 100 x human serum was injected after regeneration of the same biochip functionalized with K-One. Comparison of intensity of the signal collected on the complementary (red cross) and the non-complementary (orange cross) immobilized ssDNA and on the surface (blue cross, background) at t = 500 s after each start of injection. Dotted lines correspond to the linear forecast.

The capacity of the biochip to capture the target ssDNA is not impacted by the number of injection/regeneration cycles. Non-specific signals measured either directly at the surface or on the non-complementary ssDNA spots remain negligeable throughout the experiment. The surface chemistry is robust and assures a constant sensitivity of measurement.

3. Consecutive cycles of hybridization/denaturation in human serum diluted 20x

Further tests repeated over 39 cycles of DNA hybridization/regeneration in human serum diluted 20x were performed. Figure 5 shows raw data collected over 39 consecutive cycles. The constant detection level throughout the experiment demonstrates the surface stability.

39 Cycles Dna
Figure 5. 39 consecutive cycles of DNA hybridization/denaturation of 50 nM DNA in a human serum diluted 20x

Signal amplitude measured during the dissociation phase (500 s after the beginning of the injection of ssDNA target) is plotted as a function of consecutive injections (Figure 6). Signals from specific interactions and controls remained unchanged throughout the experiment.  Figure 5, demonstrates the presence of air bubbles on the surface at cycle 1, 16, 19, 20, 26, 27, 29 and 36, as an artefact of the fluidic system. The bubbles did not compromise subsequent measurements. Injections affected by air bubbles were omitted from the analysis in Figure 6.

Dna Injection Diluted 20x
Figure 6. Consecutives cycles of DNA injection/regeneration in human serum diluted 20x. 50 nM of the target ssDNA in human serum dil. 20x is injected after regeneration of the same biochip functionalized with K-One. Comparison between the intensity of the signal collected on the complementary (red cross) and the non-complementary (orange cross) immobilized ssDNA and on the surface (blue cross, background) at t = 500 s after each start of injection.

Comparison of the specific and non-specific interaction levels recorded in SPRi using Kimialys proprietary K-One surface chemistry in a complex biological medium (human serum diluted 20x) confirms that it generates robust biochips with maintenance of constant chip sensitivity, regardless of the number of cycles and analyzed interactions.

Conclusion

Repetitive cycles of specific interactions (DNA hybridization) were carried out in human serum using SPRi technology. The surfaces could be repeatedly regenerated and reused with high reproducibility as Kimialys proprietary K-One surface chemistry reduces non-specific binding on biochip surfaces, supporting high stability and reproducibility of macromolecular interactions in complex biological media, such as efficient DNA hybridization in human serum.