Instrument Presentation

Our systems are based on prism-coupled SPR on the Kretschmann configuration.

A collimated beam is sent towards the functionalized gold surface through the prism in order to illuminate the entire spotted matrix. There is no need to perform a 2D scan of the matrix.

After reflection on the chip, the beam is intercepted by the imaging sensor of the camera (2D matrix) where each pixel corresponds to a given location on the SPRi-Biochip.

HORIBA Scientific’s proprietary optical configuration enables a simple, yet very precise imaging system without anything moving other than a scanning mirror.

The rotation of this mirror allows the selection of the working angle relative to the resonance angle for kinetic measurements. The working angles (positions at which kinetic curves will be recorded) are chosen at the highest slope of the reflectivity curve.

Kinetic measurements consist of monitoring reflectivity variations against time simultaneously, on up to several hundred spots.

With classical SPR systems, it is possible to monitor only a few interactions in parallel. Imaging systems enable monitoring of up to several hundred interactions in parallel thanks to a special flow cell.

The number of channels corresponds to the number of different ligands that you can immobilize. The ligands are immobilized in the system, using the microfluidics system. Microfluidics requires less sample volume, but has limitations for some applications (serum, plasma, cells, etc.). Our fluidic configuration allows the study of crude samples without risking the clog of the flow cell or the fluidic parts.

Also, the coupling with other techniques, such as mass spectrometry, is more complicated with channel systems. In fact, the sample must be recovered several times and reconcentrated, increasing experimental steps, risking a sample contamination and/or a sample loss. HORIBA Scientific instruments make SPRi-MS fast and straightforward. Indeed, the design of a dedicated biochip allows identifying analytes directly on the biochip without needing a recovery step.

Comparison between multiplex and channel formats:

One working day with our SPRi instruments is equivalent to 3 working months with a channel type instrument.

High-throughput information is retrieved in a single run. For example, with a single SPRi-Biochip, it is possible to optimize the experimental conditions for the interaction (immobilization concentration, immobilization pH, immobilization buffer, etc.) and/or to screen different ligands simultaneously. Multiplexing allows you saving time and reducing consumable costs.

The imaging allows monitoring simultaneously the resonance conditions on the whole surface of the biochip thanks to the camera. It is possible to visualize all the spots of the biochip (flow cell image) and the spots where the interactions occur after the injection of the analyte (difference image).

In our configuration the ligands must be immobilized outside the SPRi Instrument with an automatic or manual spotter because we are working in array format and the fluidic configuration doesn’t allow making spots on the biochip surface inside the SPRi instrument. Whereas with the classical SPR apparatus, the ligands are injected in the SPR system and they are immobilized directly in the channels. This spotting in microarray format, allows higher throughput ligand immobilization.

Using a macrofluidics allow to analyze a complex solutions and crude samples. This opens the pathway to the exploitation of new applications and to analyze samples such as hybridoma, sera^7,8, saliva⁹, milk¹⁰…

Instead of injecting different analyte concentrations (“classical” analysis), we immobilize different ligand concentrations on the biochip surface, and we inject the analyte at one concentration (“Single injection” analysis). Dedicated software allows then determining kinetic constants and calculating the affinity of the interaction.

No because the ligands are usually covalently fixed to the SPRi-Biochip and cannot be removed. However, it is possible to inject many different samples over the same SPRi-Biochip surface, thanks to the regeneration solution.

This step means breaking the specific binding between ligand and analyte. The following solutions can be used depending on the biomolecules studied:

• Cation chelators: EDTA
• High ionic strength: NaCl 1 M, MgCl₂ 2 M
• Low pH: Glycine-HCl 100 mM pH 2, HCl 10-100 mM
• High pH: Glycine-NaOH 100 mM pH 9.5, NaOH 10-200 mM

Our SAM and 3D surfaces are designed to tolerate these regeneration solutions, and with ligands being properly anchored to the sensor surface, several successive interaction-regeneration steps are made possible. The number of regenerations depends on the affinity between the ligand and the analyte, and the robustness of the ligands.

An SPR experiment is carried out in a buffer solution. The buffer solution is continuously circulating inside the instrument. According to the ligand/analyte interaction study, the composition of the running buffer is expected to improve specific binding. Salted buffers like PBS, HEPES or citrate are recommended. Buffer composition, concentration or pH must be adjusted according to the studied biomolecules.

It is possible to detect small molecules down to about 200 Da (direct detection). However, the successful detection of a small molecule depends on the interaction model and on the experimental conditions.

Detection of small molecules (kinetic curves and affinity determination). The experiments were carried out with a CMD Biochip and the SPRi-CFM.

^{Fig. 12-13: Detection of small molecules (kinetic curves and affinity determination). The experiments were carried out with a CMD Biochip and the SPRi-CFM.}

The sample solution is injected using a classical fluidic system (no micro-fluidics); it is thus possible to inject highly concentrated sample solutions such as serum, cell lysates and plasma.

It is also possible to immobilize proteins from a crude sample on the biochip surface with a good specificity thanks to a capture antibody and an efficient cross-linking step in order to prevent the uncoupling of the protein from the biochip surface during the regeneration step. We call this spotting multistep immobilization.

The coupling between SPRi and MALDI-MS (Matrix-assisted laser desorption-ionization-Time of flight (MALDI-ToF) is very simple. It is possible to perform the two analyses on the same SPRi-slide.

Following the SPRi interaction using our systems (using MS compatible conditions, i.e. buffer, chemistry, etc…), the SPRi slide is removed from the SPRi system. Matrix deposition and enzymatic digestion occur on each individual spot. Then the SPRi slide is placed on the MALDI plate holder and mass analysis is carried out directly on the surface of the chip. No long procedure of elution which can cause material loss or sample contamination is necessary^{16,17, 18, 19}.

MALDI-MS is suited for glycans, peptide mass analysis, small or digested proteins, polymers, DNA or any organic compounds.

We can successfully characterize proteins and peptides issued from in-situ digested proteins.

Every step of the SPRi-MS experiment is performed on the same SPRi-slide surface. Indeed, the SPRi slide surface is used for ligand immobilization, specific analyte capture, insitu digestion (if necessary) and MALDI-MS analysis.