Using a high performance machine
Good quality data can only be obtained with instruments that are in perfect working order. Regular maintenance of the system by the user and the service engineer will ensure that the machine is functioning properly and providing the highest quality data possible (1).
The Control Software of the BIACORE 2000/3000 instruments includes several tools that help with the maintenance. The examples given below are for the Biacore 2000/3000 instruments but are also applicable to other types of instruments. The maintenance tools can be divided into three categories:
- mechanical maintenance
- cleaning tubing and IFC
- leaving the instrument
The following part is historically based on the Biacore 2000, but will apply to most SPR instruments.
Starting with the mechanical maintenance, the first inspection involves the syringes, which provide the flow and deliver the sample. Syringes should not contain air bubbles and they must not leak. Then, an inspection of the injection port is done. Build-up of salt may occur and this must be washed away with water. The next step is to clean the vial dislodger (the arm that holds the vials down). Clean it with a tissue with water. The final step involves the cleaning and positioning the injection needle. Wipe the needle with a tissue with water. For the best injections, the needle should go smoothly in the injection port.
Although it is not really a mechanical part, the photo-detector array that is used to determine the surface plasmon resonance angle should be checked for its performance (2). The figure ‘Reflectance dips’ shows the reflectance over a small range of pixels. The actual curve is a fitting that enables the system to measure the resonance angle with great precision. The ‘Normal Dips’ curve goes down to a reflectance of ~10.000. The second ‘Normal dips’ curve dip shifts over a few pixels because protein has bound and thus the refractive index on the surface has changed. Two abnormal dips are also depicted in the figure. Because the SPR angle is measured not at one point but in a larger section, surface heterogeneity will average the signal. This results in the shallow dip-like curve 3. A large change in refractive index (e.g. by glycerol or DMSO) can go beyond the dynamic range of the detector, which results in the absence of a dip, such as in curve 4.
A slight, shallow dip should not be a problem. Flowing buffer at a high flow rate will push out tiny air bubbles. In addition, high protein immobilization will give a slight shallow dip pattern. However, for the best performance the sensor chip surface should be homogeneous and give normal dips.
Tubing and IFC
Cleaning the tubing and IFC is started with the removal of small air bubbles and particles using the "unclogging" tool. This tool will pass running buffer at high speed through the tubing's and IFC.
The other cleaning steps use chemicals that destroy proteins and can damage the sensor chip surface. Be sure to insert a dummy-chip before performing the following cleaning steps.
Desorb removes adsorbed proteins from the IFC and auto sampler. Some measurements will deviate in the first few runs after the DESORB command due to new adsorption of proteins to the tubing. It is possible to pre-coat the tubing with BSA assuming this will not interfere with the experiments that will follow.
Sanitize cleans the pumps, tubing and IFC, removing any micro-organisms. This method uses hypochlorite solution and prolonged incubation times.
Superdesorb is a more thorough cleaning and is done as follows:
|Solution||Command||Times in a row|
|50 mM glycine-NaOH pH 9.5||Prime||3|
|Aqua dest 40°C||Prime||5|
|Solution||Command||Times in a row|
|0.5 % SDS||Prime||1|
|6 M Urea||Prime||1|
|1% acetic acid||Prime||1|
|0.2 M NaHCO3||Prime||1|
|Aqua dest 40°C||Prime||3|
The overall instrument maintenance is summarized in the table below:
|syringe inspection||once a day||2 minutes|
|injection port cleaning||once a week||5 minutes|
|vial dislodger cleaning||once a week||2 minutes|
|needle positioning||once a week||5 minutes|
|Unclogging||once a day||4 minutes|
|Desorb||once a week||22 minutes|
|Superdesorb||once a month||90 minutes|
|Sanitize||once a month||45 minutes|
Leaving the instrument
There are three tools that can be used when leaving the instrument unattended for a while:
- Append is used in the method files at the end of an experiment. It ensures that the running buffer will keep flowing for 3 or 4 days depending on the machine. Newer SPR instruments sometimes switch automatically to a standby mode with a reduced flow when idle.
- Close is used when the machine will not be used for a while but probably within five days. The procedure performs injections with water to prevent salt depositions.
- Shutdown is used when the machine will not be used for a long period. This procedure flushes the tubing and IFC with ethanol and fills them with air.
|Append (in method)||after experiment||none|
|Close||leaving for 5 days||11 minutes|
|Shutdown||leaving for more than 5 days||10 minutes|
|(from BiaUser meeting 23 march 2001 Breda - Markus Grodd)|