Biomolecular Interactions

• Protein to Protein
• Protein to DNA
• Protein to Sugar
• Protein to small molecular / drug / fragments / hormones
• Antibody interactions
• Affimers
• And many more! As long as the binding partners are soluble and there are means to couple one of them onto a sensor chip surface.

Through this technique, you can determine the strength of interactions, as well as the real time kinetics, i.e. the association and dissociation rates, which can allow you to reach new research conclusions.

SURFACE PLASMON RESONANCE

In the late 1960s, physicists Otto and Kretschmann observed a phenomenon of total internal reflection when incident polarized light hits a thin metal film at certain angles (Kretschmann and Raether, 1968; Otto, 1968). This phenomenon, known today as surface plasmon resonance (SPR), is the result of resonance when plasmons absorb the energy from the polarized light, and oscillate locally, resulting in a propagation of an evanescent wave (plasmon) perpendicular to the plane of oscillation (Figure 1).  This phenomenon proved useful for analysis of thin films and surfaces as the angle of total internal reflection is dependent on the mass on the surface of the thin film, and was used by material scientists and physicists for surface analysis for several decades. The idea and application of SPR into biosensing, detecting molecular interactions and observing the binding kinetics in real time was pioneered by Liedberg et al., in 1983. 

_{Figure 1. The SPR phenomenon
Incident light from a light source at non-SPR angles is reflected by the metal film towards the detector. However, when light is beamed at the angle where SPR occurs, energy from the light source is absorbed in resonance by the plasmons on the surface of the metal film, resulting in little or no light reflected to the detector.  An evanescent wave propagates perpendicular to the plane of electron oscillation with the amplitude of the wave diminishing exponentially with increasing distance from the surface, limiting biosensing to within 150 nm from the surface.}

In the original manuscript based on Liedberg's MSc thesis, it was demonstrated that the binding kinetics of immunoglobulin G (IgG) to anti-IgG at different concentrations could be monitored by the change in “resonance angle”, the change in angle of incidence where SPR occurs (Liedberg  et al., 1983). This allowed sensing in both liquid and gases, and the idea resulted in the eventual birth of Biacore under Pharmacia/Amersham, pioneers of SPR-based biosensors, which was finally acquired by GE Healthcare in 2008. The basis of SPR bio-sensing starts with the presence of a thin film of metal, gold, silver, titanium etc. Gold was the predominant metal of choice due to its chemical inertness, high sensitivity and good signal-to-noise ratio, and the possibility of gold-thiol bond that facilitates immobilization of molecules as self-assembled monolayers (matrix) on the gold surface, allowing useful derivatizations (Hakkinen, 2012). As pure gold itself easily adsorbs random molecules, a surface coating or matrix was necessary to block all available sites on the gold surface to prevent non-specific interactions of molecules.  At the same time, the surface coating must be selectively chemo-reactive, for the specific conjugation of ligands of interest. 

In the Biacore instruments, carboxylated methyl dextran (CMD) is the matrix of choice (Lofas, 1995). Apart from having excellent hydration/dehydration properties, chemical stability, and low non-specific bindings to proteins, CMD is structured like little bushes on the gold surface, with its long mesh-like multi-stranded structure greatly increasing the amount of ligand that can be coupled as compared to a simple alkane-thiol monolayer (Figure 2).

50 nm of gold is sputtered onto optical glass, which is then coated with a self-assembled monolayer of carboxylated methyl-dextran (CMD). Carboxyl groups present on the CMD allow for easy amine-reactive chemistry, using n-hydroxysuccinimide (NHS)/ethyl-carbo-diimide (EDC) activation and either blocking with ethanolamine or further modifying with 2-(2-pyridinyldithio) ethaneamine hydrochloride to derivatize the surface for thiol-coupling (Figure 3). Amine Coupling is a popular method with SPR biosensors and is used quite predominantly. Other coupling methods such as antibody capture, His-tag capture in NTA, streptavidin to biotin, or thiol coupling can also be utilized.

_{Figure 2. The Sensorchip Surface Chemistry}

_{Figure 3. Carboxyl groups on CMD are activated with a 1:1 mixture of ethyl-carbo-diimide (EDC) and n-hydroxysuccinimide (NHS) to form a stable amine-reactive ester on dextran before application of the ligand to be immobilized. Primary amines on the ligand react with the NHS-ester to form a covalent amide bond to the dextran matrix.}

By varying the angles of incident light on the film, it is possible to excite plasmons at a particular angle known as the SPR angle, at which light energy is absorbed and minimally reflected to a detector.  When a binding event occurs on the surface of the gold film, the change in mass brings about a change in the angle where SPR occurs. By monitoring the shift in SPR angle over time, one can observe the binding event, and the dissociation from the surface in real time Figure 4). This shift in SPR angle is commonly referred to as a response signal, and is given as Response Units (RUs) where 1000 RUs is equivalent to 1 nanogram of mass change, and a 0.1 degree shift in angle.

_{Figure 4. Real-time observation of binding through monitoring changes in SPR angle A fixed SPR angle is seen when buffer flows with nothing binding to the ligand on the surface, no mass changing. When an analyte is injected, it binds to the ligand and increases the mass on the surface, resulting in a shift in angle to the right. With increased binding, the SPR angle will shift to the right even more}

In a SPR binding assay, one of the interactants, termed as the analyte, is injected across immobilized ligand on a flow cell of a sensorchip. Binding events are observed as a rising exponential, with the curve tapering off towards equilibrium, when association and dissociation rates are equal. Equilibrium may not be achievable for low affinity interactants or low sample concentrations. At the end of analyte injection, only buffer flows across the surface with the bound analyte falling off at a rate known as a dissociation rate. Alternatively, when a co-injection method is used, a second solution, or analyte is injected in the dissociation to allow one to observe competition.  When all bound analyte has fallen off the surface, the surface is considered “regenerated” and the signal will be back to baseline ready for the next analyte injection (Figure 5).

_{Figure 5. Typical events on the surface of a sensorchip
Both association and dissociation events on the surface are monitored in real time.}

In our SPR instruments such as the Biacore T200 and the Biacore 3000, the built-in microfluidic system creates on the carboxymethylated surface of a sensorchip four flow cell channels in serial flow design. For typical binding experiments, active referencing is achieved when two channels are used and samples are serially injected across the reference flow cell followed by the ligand flow cell. The sensorgrams obtained from both flow cells are then subtracted to give the specific binding curves that describe the kinetic parameters of the interaction studied. With the advanced microfluidics for sample delivery on the Biacore T200 / 3000, in the dissociation phase one can choose whether to have running buffer or a different buffer/solution/sample injected immediately in this phase by selecting the appropriate injection methods (Figure 6).

_{Figure 6. Active referencing of both normal injection and co-injection methods
Serial injection across reference surfaces before the ligand flow cell. In a typical scenario, where running buffer is matched with the sample buffer, upon injection of the analyte without non-specific bindings occuring, the reference flow cell sensorgram is flat or a small box shape, whereas in the ligand flow cell, the binding curve is seen. The two curves can then be subtracted to give a final referenced subtracted sensorgram.}

Typical serial flow path of Biacore Instruments, where there are 4 flow channels, with the first flow cell being commonly used as a reference to subtract out refractive index difference and non-specific bindings, and the other three flow cells onto which different ligands can be immobilized. The same analyte that goes across flow cell 1, will go across flow cell 2, 3, and 4 serially, ensuring that data collected is unbiased with the exact same sample in contact (Figure 7). 

_{Figure 7. The Serial flow path of a typical Biacore 4 channel sensorchip}

SPR AT NTU

The first paper in history where SPR was used in a biosensing application appeared in Sensors & Actuators in 1983, where Liedberg et al. demonstrated the monitoring of an antibody to antigen interaction in real time.

Several years after, commercial biosensing instruments built around this principle, together with excellent surface chemistries and microfluidics, allowed many end users to characterize the kinetics of the interactions they were interested in. 

To date, at least 10,000 publications contain SPR biosensor data, which averages to approximately one publication a day for the past 30 years. 

In 2007, an SPR Biosensing platform was established in Singapore by Associate Professor Susana Geifman at the School of Biological Sciences, NTU, serving the SPR biosensing needs of NTU and other institutions. Testimony to the importance and success of the technology, the SPR Biosensing platform had successfully carried out hundreds of collaborations, projects and contributed to many publications. Data from SPR had also help PIs strategize and refine their research finding/directions. 

SPR based biosensors are now indispensible in both scientific research, clinical settings, and pharmaceutical industries for numerous applications they serve, from validation of drug bindings, characterization of molecular interactions, to clinical diagnostics and many more. Instrumentations have also advanced tremendously in terms of automation, sensitivity and functionalities. In future, SPR based biosensors will remain highly relevant, and will continue to serve the needs of research and public health. 

In 2009, Professor Bo Liedberg from Linköping University, Sweden, came to NTU on a Visiting Professorship and joined NTU as a full faculty in 2012. He established the Centre for Biomimetic Sensor Science in NTU, together with the SPR platform at SBS, many high quality, international molecular interaction workshops and events had been organized, such as the Advanced Biosensor Workshop in 2011, the 30 Years of SPR Biosensing symposium in 2013, etc. 

The SPR Biosensing platform is currently integrated into the BRC's Biomolecular Interactions (BMI) platform, and is open to NTU and non-NTU users.

Circular Dichroism is the differential absorption between left and right circularly polarized light in passing through a sample. 

• Approximately secondary structure content, % of alpha helice, beta sheet, random coils etc
• Comparing between mutant to wild type proteins to see if there is any structural difference
• Comparing between preps to ensure protein is "folded" similarly between samples
• Temperature Ramping to unfold a protein with increasing temperature, observing how the structure content changes
• Conformational changes in the presence of ligands or drugs and many more! 

The Malvern Zetasizer ZS-Nano

The Zetasizer Nano ZS is a very quick, and extremely useful instrument to learn and use. The NanoZS model allows us to size molecules of diameter between 0.3nm and 10 microns. Whether it is proteins, or bacterial cells in solution, the NanoZS allows one to rapidly obtain information on the population distribution of sizes within a sample, with sample being recoverable. Typical disposable cuvettes will require approx 400ul of sample. 

One can use this technique to determine protein sample homogeneity, oligomerization studies, sample quality over time, and even use it for protein "melting" with its heating function up to over 95 degrees celsius.'

Zetapotentials, the velocity/speed of particles in solution when subjected to an electric field can also be measured on this instrument. 

Malvern iTC200

Isothermal Titration Calorimetry (ITC) is used to measure reactions between biomolecules. The methodology allows quantitative determination of the binding affinity, stoichiometry, entropy and enthalpy of the binding reaction in solution, without the need to use labels or to immobilize molecules.

When binding occurs, heat is either absorbed or released and this is measured by the sensitive calorimeter during gradual titration of the ligand into the sample cell containing the biomolecule of interest. 

• Quantify binding affinity
• Candidate selection and optimization
• Measurement of thermodynamics and active concentration
• Characterization of mechanism of action
• Confirmation of intended binding targets in small molecule drug discovery
• Determination of binding specificity and stoichiometry
• Validation of IC50 and EC50 values during hit-to-lead
• Measurement of enzyme kinetics