معطل الموجات فوق الصوتية القابل للبرمجة: بناء سير عمل صوتي قابل للتكرار

When the laboratory requires more control than is available with a handheld homogenizer, a programmable ultrasonic disruptor might be the answer. Can disrupt cells, disperse particles, homogenize suspensions, shear nucleic acids or sample preparation for further analysis.

The instrument is easy to use. The challenge is to find conditions that will process the sample properly with no damage occurring to the material that the laboratory wants to recover, and with no overheating of the sample.

This is where programmable control comes in handy. The operator can set the amplitude and/or the processing time, the pulse cycle, the limit temperature or the total energy without holding a button and without judging the result by sound. After an optimized method has been established, the optimized conditions can be repeated for subsequent samples.

How Ultrasonic Disruption Works

The ultrasonic disruptor is an apparatus that transforms the electric power into mechanical vibration. That vibration is passed to a probe which is placed in the liquid sample by the converter.

When the probe moves at high speed in the liquid, pressure changes are generated which cause the formation of microscopic bubbles of liquid. These bubbles grow and shrink and are called cavitation. The forces generated in the local area may cause cell membranes to be disrupted, aggregates to be broken apart and allow for better mixing or dispersion.

In terms of practical use, the probe is inflicting a tremendous amount of mechanical energy on a small area. This is why probe sonication can be quick, but can also be detrimental to a sensitive target, or cause foam in an experiment.

Why Programmable Control Matters

Two samples may be treated for the same length of time, but not receive the same treatment.

The movement of ultrasonic energy through the liquid will be affected by: sample volume, viscosity, temperature, probe diameter, immersion depth and container shape. The programmable instrument enables the operator to control the variables which can be realistically standardized.

The modern ultrasonic processors can have adjustable amplitude, timed operation, pulse mode, energy display, energy setpoints and temperature monitoring.

These functions are not only convenient but useful as well. They make a method more easily documented and explain exactly how a sample was processed.

The following: “sonicated for one minute.” is an inadequate method. It should specify the type of probe, setting for the amplitude, the pulse cycle, the total processing time, the sample volume, the method of temperature control, and the container type.

Use a starting amplitude, not a maximum wattage

Probably the biggest error is to think that more watts = more power into each sample.

The amplitude is a measure of the tip motion of the probe, and is more directly associated with sonication intensity. Power used by the instrument will vary depending upon the resistance that is generated by the sample, the probe, the viscosity and operating conditions. When a method needs to be reproduced, it is important to keep the amplitude, temperature, viscosity and sample volume the same, Sonics says.

When developing methods, start with a moderate amplitude and don't use the highest amplitude at first. Run a small test sample, look at the result and only increase the intensity of the process if required.

Excessive sonication could lead to better disruption of cells, but also cause higher temperature, foaming, wear of the probe and damage to sensitive proteins or cellular structures.

The appropriate amplitude is thus dependent upon the application. Cell lysis and/or dispersion of nanoparticles, preparation of organelles and/or shearing of DNA should not be expected to be the same.

Control heat with Pulse Mode

With continuous sonication the temperature of the sample can be increased rapidly. This is of importance when using proteins, enzymes, membrane structures or other temperature sensitive materials.

Mechanical methods of cell disruption can generate localised heating, which can result in protein denaturation or aggregation. Therefore preservation of the sample's quality is important if the protein quality should be preserved, and for this purpose the sample should be kept chilled.

Pulse mode is a cycling between sonication and relaxation. In an off cycle the sample is given time to cool and settle around the probe prior to the next burst. Intermittent operation is also mentioned in the equipment manuals to help to lower the heat buildup and therewith to increase the consistency in the processing.

Pulse mode won't eliminate the need for temperature control. Even though the tubes are small, they can heat up quickly, particularly at high amplitude. It may still be necessary to use an ice bath, chilled holder, a temperature probe or a programmed temperature cutoff.

Ensure that the Probe matches the Sample Volume

There is a significant influence from the type of probe used.

A little microtip gathers power in a small element which can be of high intensity at a small spot. The more power you will have the more material the larger the probe. As seen from the manufacturer's instructions, probe diameter and actual processing volume are highly correlated but the end range of values is dependent on the process.

A probe that is too large for a small sample can splash and/or over heat. A small tip in a large vessel could cause the uneven processing and have an unnecessarily long processing time.

The probe should be sufficiently deep to efficiently transfer energy into the liquid but not so deep that it sucks too much air into the liquid sample. During the vibration it should not come in contact with the bottom or the side of the container.

The shape of the containers is also important. If a narrow vessel is used to focus energy, it can focus energy differently than a wide beaker and using a different tube or vessel in the same study can change the result, even if the instrument settings are the same.

Construct the Method Around the Sample – CSTA 2014, Section 2, Grade 12, Strand B, Task 3

Typically, a soundable method for sonication is found after a few brief trials.

Start with the real sample type, the sample size, concentration, buffer and container intended to be used for routine analyses. Must keep the sample cold; select an appropriate probe. Use medium amplitude and brief pulses.

Review the outcome of each trial using a method which simulates the actual goal. This can be microscopy, protein yield, enzyme activity, viscosity and/or clarification following centrifugation. It may include particle-size measurement and/or visual stability for particle dispersion.

Don't assess success based on the level of turbidity or clarity of the sample. The activity loss of the target protein may have occurred before the appearance of the sample.

After acceptable conditions are determined, carefully document the conditions and repeat the test with a number of independent samples. There's no better way to test the reliability of a method than to have it repeat itself.

Common materials used in the laboratory

This kind of programmable ultrasonic disruptors are widely adopted in the following fields:

• The disruption of bacteria and mammals cell wall.The breakdown of bacterial and mammalian cell walls.
• The preliminary cutting of tissues needs to be followed by tissue homogenization.Tissues should be homogenized after preliminary cutting.
• This covers the procedures necessary to prepare protein and nucleic acid samples.The sample preparation of protein and nucleic acids.
• Chromatin shearing by DNA or chromatin shearing.Shearing of DNA or chromatin.
• A dispersion of particles and nanoparticles.
• Emulsification
• Deagglomeration
• Extraction and mixing
• Difficulty in preparing suspensions.Difficulty in making difficult suspensions.

There are several different physical methods that can be used for cell lysis, such as sonication. The most suitable approach will vary with the cell type, sample size, downstream application and sensitivity of the material to be extracted.

If a low intensity processing or other disruption technique is better suited for delicate organelles or easily-destroyed proteins, use that technique.

There are common operating problems that you should avoid

The foaming is usually caused by the probe being too close to the surface of the liquid and/or the sample being processed too intensely. A slight descent of the probe, a decrease in amplitude or a change of the pulse cycle can be helpful.

The reasons for poor disruption are that the amplitude is too low, the wrong probe is used, too much sample is used, the sample is not immersed the right depth, or too short of a total processing time.

Sometimes a number of variables are changed to make the results inconsistent. The method can vary depending on the tube used, the starting temperature of the sample, viscosity, depth of penetration of the probe into the sample, and volume.

When a mechanical noise is heard or is odd, it should not be ignored. It can be a sign of loose probe, damaged probe tip or a misassembly. The instrument should be stopped and checked before further processing should be done.

Probe tips are also eroded by cavitation erosion. If the surface is damaged, then the transfer of energy will be effected and metal particles can be introduced into the sample. Check the probe frequently and replace as per manufacturer's instructions.

Work Safely

Use proper stand and clamp to secure the converter, do not make an unstable assembly.

However, ultrasound processors will make an audible sound and the manufacturer's guidelines suggest hearing protection when in use. A sound enclosure will provide a reduction in exposure and, also, contain splashes or aerosols.

Properly handle and contain biological material and follow laboratory safety procedures. Control of the probe depth should be made to minimise aerosol formation and foaming. Rinse between samples and decontaminate the probe in an appropriate rinse procedure that is effective for both the probe and the sample.

استنتاج

The result is affected by the amplitude, the pulse cycle, the size of the probe, the volume of the sample, the temperature, the depth of the sample into the container, and the shape of the container. The hardest pace is not a good place to begin.

A reliable workflow starts with brief, well-controlled trials, then it passes to a proper downstream test to assess the outcome, and then sufficient information is collected so that it can be repeated by another operator.

الأسئلة الشائعة

Q1. What is Programmable Ultrasonic Disruptor used for?

For cell lysis, homogenisation, dispersion of particles, shearing DNA, emulsion, extraction and various applications for liquid processing.

Q2. Better Higher Amplitude?

No. An increase in amplitude will increase the intensity of the sonication, however may result in increased heating, foaming, probe wear and sample damage.

Q3. Why Pulse Mode is used?

Pulse mode: Periods of rest between the ultrasonic bursts. This will help to reduce the heat accumulation and return the material back around the probe.

Q4. What is the optimum size of the probe?

The volume, size of the sample vessel and intensity of processing required should all be matched by the probe. Small tips are good for small volumes and large probes are good for large volumes.

Q5. How to Make the Results of the Sonication Reproducible?

Probe, amplitude, pulse cycle, volume, temperature, immersion depth, sample composition.