Before analysis, samples must be prepared so they can be observed by the instrument properly. There are a variety of methods for preparing samples for different applications, from complex processes like microwave digestion to simpler processes like milling and liquid handling. Automated sample preparation systems have numerous benefits, such as increasing lab throughput, lessening the margin for error, and minimizing the risk of staff developing repetitive strain injuries.
In this eBook, you’ll learn about:
- Questions to ask when purchasing a sample preparation system
- Reducing lab plastic and reagent waste
- Advances in mass spectrometry-based proteomics
- The benefits of automated liquid handling for microscale samples
- Getting the most out of your homogenizer
- How to achieve consistency with microwave digestion
? Questions to Ask When Purchasing a Sample Preparation System
? Reducing Lab Plastic and Reagent Waste
? Advances in Mass Spectrometry- Based Proteomics
? The Benefits of Automated Liquid Handling for Microscale Samples
? Getting the Most out of Your Homogenizer
? How to Achieve Consistency with Microwave Digestion
SAMPLE PREPARATION
RESOURCE GUIDE
Questions to Ask When Purchasing a Sample Preparation System
by Lab Manager
Before analysis, samples must be prepared so they can be observed by the instrument properly. There are a variety of methods for preparing samples for different applications,
from complex processes like microwave digestion to simpler processes like milling and liquid handling. Automated sample preparation systems have numerous benefits, such as increas- ing lab throughput, lessening the margin for error, and mini- mizing the risk of staff developing repetitive strain injuries.
5 Questions to Ask When Buying a Sample Preparation System:
1. How much time is your staff spending preparing samples manually? Spending too much time doing repetitive tasks can lead to repetitive strain injury. Automated sample prep systems could alleviate the risk.
2. Are your staff having trouble keeping up with daily sample throughput?
3. Do you have a consistent throughput of samples prepared the same way every day?
4. Which step in sample preparation would be easiest to automate?
5. Are more complex, higher-value projects being put on hold because your lab staff does not have the capacity to pursue them?
Selecting the Right Particle Reduction Method
There are several methods of particle size reduction suitable for different types of samples. For hard, brittle, dry samples, look for a grinder that employs pressure, impact, or friction grinding. Soft, fibrous, elastic samples should be prepared with shearing or cutting grinders.
Reducing Lab Plastic and Reagent Waste
Advanced technology, reuse, and reduction can be combined to make labs more sustainable
by Mike May, PhD
Science labs produce gigantic volumes of waste that are often far from being environmentally friendly. In particular, sample preparation can be a crucial creator of plastic and reagent waste.
No one knows just how much waste is created specifically by sample prep, but it’s likely increasing. This is especially true for biological analyses such as molecular workflows.
To address these issues, scientists must create a similar scale in the battle to reduce plastic and reagent waste without sacrificing quality.
Unfortunately, no one-approach-fits-all solution exists for reducing waste in sample prep.
Starting at the source
When it comes to plastics used in sample prep, better solu- tions start at the source. Lab-grade, single-use plastics are made with fossil fuels that can be biohazardous and are also non-biodegradable as waste.
One improvement could come from a more environmentally- friendly source material. For example, some manufacturers
now offer things like pipette tips made from largely biobased plastics. Alternatively, scientists can pick reusable products.
In some situations, sample prep might even be reworked
to cut out the need for plastic. Experts believe that acoustic handlers that skip tips altogether reduce plastic in high- throughput scenarios.
When most scientists think of more sustainable uses for pla- stic, recycling probably comes to mind. This sounds like a great idea in theory, but it’s largely failed in practice. Experts state that currently only 16 percent of plastics waste is repro- cessed to make new plastics.
Despite the dismal data, experts recommend that working with a company that maintains a closed-loop system for recy- cling and reusing plastics and looking for products made from compostable polymers may make a difference.
Reducing reagents
Beyond the use of plastic, sample prep usually requires various reagents, which can be just as problematic. Many reagents become toxic chemical-waste streams, and dispos- ing of them is costly and carbon-intensive.
The easiest way to reduce reagent waste is to use less. Material volumes per sample can be reduced upfront. Mini- aturizing protocols, by moving lower throughput bench preps traditionally performed in microtubes to multi-well plates, provides one approach.
Some manufacturers offer sample-prep platforms that re- duce the need for reagents. For instance, the use of microflu- idic-based devices requires far lower levels of reagents than conventional methods.
Even if the volume of reagents required can’t be reduced, the chemicals can be greener. To delve deeper into this top- ic, chemist Elefteria Psillakis of the Technical University of Crete in Greece and her colleagues described 10 principles of green sample preparation. As part of this strategy, Psil- lakis and her colleagues encouraged “eliminating, replacing, or minimizing harmful solvents and reagents.”
Pay attention to the packaging
Beyond the plastic tips, reagents, and supplies that a lab buys, the packaging makes up a key component of the waste. Labs can reduce that packaging in various ways. First, a lab manager can consolidate orders for supplies.
In addition, labs are encouraged to work with manufacturers that have take-back programs for items, such as pipette-tip boxes and packaging.
When scientists pay attention to the packaging, the impact can go beyond a lab. As a result, manufacturers might im- prove their packaging.
One by one
The expanse of the challenge in reducing sample prep waste may seem daunting, but individual labs can make a difference. As an example, marine chemist Jane Kilcoyne and her col-
leagues at the Marine Institute in Ireland made a dedicated effort to reduce lab waste. As they reported: “Methods were verified to allow transitions to more sustainable and envi- ronmentally-friendly consumables, replacing plastics with paperboard and glass alternatives, leading to a reduction in the consumption of single-use plastics by 69 percent.” They added: “Adoption of green analytical chemistry principles to procurement and preparation of chemical solutions led to a reduction in hazardous chemical waste by ~23 percent.”
Getting started with sustainability
Given this data on waste from sample prep, where does a lab begin to make reductions? Experts recommend the use of waste audits to quantify the type and quantity of waste. The lab can then act on that data with guidance from resources like the Sustainable European Laboratories Network and My Green Lab.
Getting your organization’s health, safety, and environmen- tal teams involved can also spread the goal of sustainability. Any improvements that a lab can make in reducing waste can make a difference.
Advances in Mass
Spectrometry- Based Proteomics
New technologies strive to enhance throughput, quantitation, and data quality
by Damon Anderson, PhD
The impact of mass spectrometry (MS) based proteomics on cell biological understanding is gaining new ground each day. Several recent advances in research and product development are now helping drive the field into the exciting new world of single-cell proteomics and beyond.
The promise of single-cell proteomics
Proteomics involves the characterization of the full com- plement of proteins within cells. Such insight can shed light on cellular processes, cell states, the appearance of disease markers, and other important details. The growing field of single-cell proteomics (sc-proteomics) narrows the focus toward biological activities and functions within individual cells. While proteomics aims to characterize protein levels in cells at the population level, sc-proteomics strives to uncover distinct qualitative and quantitative differences among indi- vidual cell types and states. This promises to inform a higher
understanding of biological function, dysfunction, and the effects of therapeutic intervention.
Scaling down proteomics
Much of the proteomics successes thus far have involved pop- ulations of cells. There are significant challenges, however, in scaling proteomics investigations from cell population levels down to the single-cell level.
Technologies such as multi-parameter fluorescence-assisted cell sorting (FACS), originally developed for the cell culture field, have been useful in the proteomic analysis of small groups of cells. Other technologies such as single-cell mass cytometry (CyTOF) have shown the capability of detecting small numbers of proteins in single cells. Although valuable, these approaches have proven to be limited in sensitivity, pro- teome coverage, and the throughput needed to analyze single cells and replicates on a practical time scale.
Mass spectrometry single-cell (sc) proteomics
Advanced MS technologies have emerged that address these limitations. New instruments are capable of attomole (10-18) sensitivity, within the range of single-cell protein levels, detecting thousands of proteins in a single run using multi- plexed sample analysis.
Sample preparation
As an important initial step in MS analysis, sample prepara- tion techniques must provide sufficient starting materials to account for losses due to handling, absorption, and back- ground interferences in samples. Separation performance is another critical parameter required to remove background interferences and optimize peptide isolation and detection. These considerations are especially relevant to sc-proteomics
and the need to identify distinct differences within small-vol- ume samples.
Sample enrichment
A growing number of novel sample enrichment technolo- gies, such as that originally pioneered by Claudia Ctortecka, Ph.D., and colleagues at the Research Institute of Molecular Pathology in Vienna, Austria, use automated single-cell iso- lation coupled with picoliter dispensing workflows. Samples are deposited on microscope slide-sized chips, typically in multi-array wells. After enrichment and labeling, samples are then prepped for MS analysis. These novel devices eliminate manual handling and evaporation, allowing direct injection of single-cell samples via a standard LC-MS/MS autosam- pler. Automating the sample handling process increases reproducibility, while the multiplexing of sample enrich- ment enables analysis of up to hundreds of single cells and quantification of thousands of proteins in a single analysis.
Low-flow nano LC
As mentioned, separation performance is vital in removing background interferences and allowing clarity and repro- ducibility in downstream analysis. New nanoflow UHPLC technologies are designed to deliver the low flow rates (10 nL/ min) and the high pressures (up to 1500 bar or more) required by the latest high-resolution columns. By eliminating dead volumes and optimizing flow, these new nanocolumns can achieve high-sensitivity detection of peptides and proteins that are present at vanishingly low levels, such as those pres- ent in sc-proteomics samples. A range of column chemistries and scaffolds, such as polymeric and nanofabricated sub- strates, enable column scouting, high-fidelity separations,
and deep sc-proteome coverage.
Protein quantitation
Quantitation is another challenge in single-cell analysis, par- ticularly for scaling up and multiplexing sc-proteomic sample replicates. To this end, the field has leveraged the use of isobaric tags. These are molecules that can be covalently at- tached to peptides to produce identical masses that fragment inside the mass spectrometer to produce different-sized ions. This technique allows peptides to be tagged without changing their initial masses and subsequently quantified upon LC-
MS/MS fragmentation. Commercialized as TMTs (short for tandem mass tags), the number of possible tagging combina- tions was recently expanded to 18, allowing multiplexing for three replicates of six samples in a single experiment.
Data acquisition and analysis
Data acquisition and data quality are big challenges for
sc-proteomic investigations as well. MS systems must deliver sensitivity and accuracy on a per-sample basis while deliv- ering the precision and throughput necessary to analyze multiple sample replicates.
These challenges are being met by the latest generation of hybrid mass spectrometers and data analysis technologies. Traditional data-dependent acquisition (DDA) of ultra-low input samples suffers from the accumulation of missing values (missing peptides) as the size of the sample cohort increases. Data-independent acquisition (DIA) methods are not fully compatible with isotope-encoded (TMT) sample multiplex- ing. Novel approaches are implementing concepts like iden- tification-independent data analysis tools to compare TMT proteome signatures across hundreds of samples and different cell types. These approaches are working to increase quanti- tative protein coverage while enhancing data reproducibility.
Outlook
Advances in technology and product development are paving the way to better accuracy, precision, throughput, and overall quality of MS-based sc-proteomics investigations. New MS instruments, in combination with the latest quantitative labels and analysis techniques, have the power to analyze over 300 cells per day, cutting the total analysis time of 10,000 cells to just over one month.
With this growing cache of advancements and accelerat- ed workflows, the focus can now broaden to include new territory. MS-based spatial-omics aims to use sc-proteom- ics to deduce discrete differences in intact tissue. Although
laser-capture microdissection and other techniques coupled with mass spectrometry have been developed, none have achieved single-cell resolution. Single-cell resolution of intact tissue is a new frontier for the field of MS-based sc-proteom- ics and it’s now within view.
Product Spotlight
Sartoclear Dynamics® Lab: One-step Mammalian Cell Culture Harvesting
Sartoclear Dynamics® Lab kits are designed for rapid harvesting of 15 mL to 1,000 mL volumes of cell cultures in the lab, enabling clarification and sterile filtration to be performed in one step. These kits simplify the process by fully eliminating the centrifugation step otherwise needed for clarification. As a result, up to 1,000 mL cell cultures can be efficiently clarified and sterilized in minutes – quickly and easily.
The Benefits of Automated
Liquid Handling for Microscale Samples
Automating sample handling can fill a growing need for applications like DNA sequencing, protein expression, biological assays, and more
by Kelsey A. Morrison, PhD
To many, the thought of handling microscale samples evokes an image of tedious manual pipetting. This time-consuming task can be largely replaced with automated manipulation of small samples. Automating sample handling can fill a growing need for applications like DNA sequencing, protein expres- sion, biological assays, and the rapid development of synthetic products.
Despite the initial monetary investment necessary to acquire these systems, automated sample handling brings distinct advantages. Laboratories working with small samples by hand face worker fatigue, reduced precision, and limitations on experimental throughput. In contrast, investing in automation can bring obvious benefits, from reduced repetitive motion injuries to greater reproducibility, and increased processing bandwidth. Additional benefits include savings from fewer wasted samples and reagents, as well as streamlined work- flows. The capability to combine sample preparation with analytical instrumentation for fully automated synthesis and analysis is another advantage.
Common types of systems for handling small-scale samples
Likely the most recognizable form of automatic sample han- dling, pipette-based systems act as robotic pipetting platforms by dispensing solutions from tips by contacting the deposition target. These pipette-based systems typically operate through either an air-cushion design for sample manipulation or with positive displacement via pistons. For applications requir-
ing higher accuracy and precision of low-volume samples, positive displacement is preferable over the lower cost, lower precision air-cushion mechanism.
Similar to the pipette-based sample handling systems are those based on syringes and pins, both of which require contact between the dispensing device and the intended end surface or solution. All three forms of contact-based liq-
uid manipulation platforms have the potential drawback of cross-contamination.
For laboratories that can afford to invest in an automated sample handling platform based on mechanisms other than pipetting, syringes, or pin dispensing, the alternatives may be better options when high precision and accuracy are para- mount, if low- and sub-nanoliter samples are to be processed, or cross-contamination is a concern. A direct comparison of results based upon data collected from samples handled in
a tip-based system and an acoustic droplet ejection (ADE) platform found statistically different results between both datasets, with the ADE system appearing to provide more consistent values. ADE sample handling is also useful for rapid, microscale synthetic prototyping, which is how it was applied for automatic reaction scouting of isoquinoline synthetic building blocks in nanoliter droplets. Microscale
acoustic manipulation has a wide range of potential applica- tions because of its precise control, short dispensing time, and compatibility with high-capacity sample wells rendering the mechanism particularly appealing in bioassays.
Getting the Most Out of Your Homogenizer
To get the most out of a lab homogenizer, it is crucial to select the right features for given applications and to maintain it properly
by Erica Tennenhouse, PhD
Homogenizers are used to disperse, emulsify, lyse, or mill samples as diverse as tissue, soil, and cosmetics. Although a simple mortar and pestle can accomplish somewhat similar results, a high level of consistency can be achieved only by a mechanical homogenizer with a decent generator probe. To get the most out of a lab homogenizer, it is crucial to select the right features for given applications and to main- tain it properly.
Picking your unit
Homogenizers come in a variety of configurations, including handheld and bench-mounted models with a variety of probes to match the correct sample type. The primary selection cri- terion for a homogenizer is the sample or material type being processed.
The wattage required will depend on both the sample type being processed and the size of the samples. Processing small volumes requires less power, so a handheld unit is usually the best choice in these cases. But to homogenize a large volume of viscous material, a larger motor may be needed. Certain homogenizer units are capable of processing a wide range of sample volumes, from milliliters to liters.
In many cases, the speed of the process matters as well. For example, faster processing time increases the quality of the yield for nucleic acids and proteins because it gets them into the protective buffer faster. Speed controls are available in some mechanical units to enable the user to adjust the speed in increments of hundreds of RPM.
The friction generated by a homogenizer will cause an increase in temperature during operation. The simplest way to reduce the effect of temperature on the samples is to select the correct generator probe and unit for your application, which will decrease the time spent homogenizing the sample, thus reducing the effect of temperature. Many manufacturers, including those of blender-style homogenizers, have also be- gun creating built-in cooling systems to combat temperature increases. Here are some of the most important features to look for when purchasing a homogenizer:
• Easy cleaning of product-contact surfaces
• A low motor noise, since homogenizers are usually located on workbenches in proximity to operators
• Ease of use
• Rapid homogenization
• User control over homogenization parameters through a familiar digital display
• Low heat generation, which is particularly important for labile tissue samples
• A programmable library of methods
Maintenance is key
Once a homogenizer has been selected and purchased, the next challenge is to keep it in good condition. The life of a
probe-based homogenizer can be extended with some simple care of the generator probe—the part that goes into the sample. Although some homogenizers do not have a generator that can be easily taken apart, purchasing a system that allows for a thorough cleaning once in a while is generally consid- ered a better investment.
The upper and lower bearings need to be replaced on a
semi-regular basis; the specific replacement time depends on their use. While a new generator probe can run more than
$1,000, a pack of bearings costs a mere $30 to $40. Thus, small repairs can provide a substantial return on investment in the long run.
Signs that it’s time to perform maintenance on your homog- enizer include the sample heating up, the generator probe or motor unit becoming hot to the touch, or black residue appearing in your sample. Wearing or discoloration on the
internal components, such as PTFE bearings, or your gener- ator probe seizing up are also signs that it’s time for service. Additionally, if the generator probe has never been taken apart for a cleaning, then chances are that some maintenance is required.
Product Spotlight
Reliable and efficient solutions for homogenizing various types of samples
PRO Scientific homogenizer packages are ideal for sample preparation and include everything needed to effectively homogenize samples, including a high- quality homogenizer, precision-designed generator probes capable of breaking samples down to a submicron level, and other accessories.
Homogenizing package kits are simplified into four categories and selection is easily done based on container/tubes:
Micro Homogenizer: Ideal for homogenizing in microtubes from 0.5-2ml and 5ml tubes
Multi-Sample Homogenizer: For high-volume homogenizing in tubes from 1.5ml - 50ml
Universal Homogenizer: Full range homogenizer for homogenizing in tubes from 1.5ml - 50ml
MaX Homogenizer: For homogenizing in larger tubes (50ml) up to beakers (5L)
PRO homogenizers are designed for ease of use, with intuitive controls and variable speed settings to allow for precise control over the homogenization process.
Additionally, PRO Scientific homogenizer probes are durable and built to withstand heavy use, ensuring reliable performance over time.
How to Achieve Consistency with Microwave Digestion
Achieving digestion consistency requires that samples be homogeneous and representative of the original sample
by Angelo DePalma, PhD
Microwave digestion works by exciting nearby water mole- cules to tear sample materials apart. Adding strong acids, or even a base, speeds up sample homogenization. What results is a mixture of organic materials at various stages of decom- position, and highly solubilized metal ions with uniform oxidation states suitable for analysis by inductively coupled plasma, atomic absorption, or atomic emission spectroscopy.
Scientists prefer microwaves because competing methods all have serious drawbacks. For example, ashing—where
samples are burnt until only ash remains—is prone to analyte loss due to incomplete combustion. Fusion decomposition, a high-temperature technique that uses salt fluxes to solubilize samples, is labor-intensive and suffers from interferences from fusion agents.
Microwave digestion solubilizes a broad variety of samples relevant to many industries, including agriculture/foods, clinical/ life sciences, environmental, geoscience and mining, metallurgy, pharmaceuticals/nutraceuticals, paints and coatings, plastics, and polymers. Materials that would not be
suitable for microwave digestion include those that would oxidize violently when exposed to acids such as explosives, propellants, and perchlorates.
Acid selection is probably the most important factor in micro- wave digestion. Performing microwave digestion in a closed vessel allows you to heat the acid above its boiling point. This increase in temperature dramatically increases the oxidation potential of the acid, allowing safer acids such as nitric acid to be used instead of more harmful acids like perchloric acid.
Nitric acid is most commonly employed for organic samples, including plant and animal tissues, oils, polymers, and phar- maceuticals. Sulfuric acid may be required to break up aro- matic hydrocarbons. For organic samples, experts recommend pre-digesting for up to 15 minutes. Pre-digestion involves adding acid to the sample but leaving the vessel uncapped, in the fume hood, so that if the sample is prone to produce a lot of gas it can discharge this gas before the vessel is sealed.
Inorganic samples do not contain much carbon and there- fore do not usually produce high pressures during digestion. They may, however, require higher temperatures than organic samples.
Power and time
When microwave digestion was first introduced, control over process parameters was limited to time and power. This can be likened to microwaving a frozen burrito. You can choose the amount of time and the power level, but you won’t know if the center is cooked or frozen until you cut into it. This was a problem for early microwave reactors, too, which led to the development of temperature-controlled units that have become the standard for digestion today.
By utilizing either contactless or in situ temperature mea- surements, the digester applies the appropriate amount of
power necessary to achieve the temperature set point, in the allotted amount of time. This method is much safer and more efficient than simple power and time control. A temperature sensor provides feedback for power levels from the sample temperature, without applying too much power and risking damaging the microwave vessels or applying too little power and not fully digesting the sample. Computer control mon- itors each reaction (in multi-sample digesters) and records relevant parameters at every stage of the digestion, thereby providing consistent output for every sample in every run.
Achieving consistency
Achieving digestion consistency requires that samples be homogeneous and representative of the original sample. Reducing the sample’s particle size before digestion using milling or grinding can improve the contact between the acid and the sample. In some cases, samples may need to be heated to promote mixing.
Digestion vessel geometry and materials of construction af- fect the efficiency of microwave digestions. Sample cup geom- etry may affect both digestion efficiency and sample recovery. Reduced-volume vessels, for example, may be a good choice for expensive or scarce materials, or for workflows where users seek to minimize risks associated with hot, pressurized hazardous materials. Lab managers who are unfamiliar with microwave digestion enjoy a range of resources for achieving consistency. The best place to look first is the application page for your microwave system vendor. Most vendors also offer training courses and will advise users on specific samples and likely safety issues.
Purchasing decisions
Labs should consider the suitability of the microwave system to sample type, daily throughput and workflow, and applica- tion support. Manufacturers offer many different vessel types and geometries, and these must be matched to sample and
throughput as well. Higher-throughput vessels, appropriate for many EPA methods, allow the digestion of large batches at moderate temperatures and pressures. However, many
inorganic samples and difficult organic samples like heavy oils and plastics require higher temperatures or pressures that are not suitable for this type of vessel. Therefore, a higher-per- formance vessel is required for these applications to digest samples completely. For this reason, you should review the samples you intend to run in the microwave before purchas- ing to save yourself a lot of money.
Daily throughput will dictate whether a sequential microwave or batch microwave offers the best workflow option for a par- ticular lab. If your lab is digesting a lot of samples every day,
a batch microwave that can digest 24 to 40 samples at a time will help you achieve better throughput. On the other hand, if your laboratory is only preparing a few samples of varying sample types, a sequential system might be a better option as it allows you to run any combination of samples and acids in its 24-position autosampler.
In sequential systems, samples enter the microwave cavity with the help of a robotic arm and every sample is precisely controlled. This gives laboratories tremendous flexibility to prepare a wider range of samples. Each sample is then digest- ed, cooled, and placed back into the rack in about 10 minutes, or a full batch of 24 samples is digested in about four hours. Operation is fully walk-away.
Sartorius is one of the world’s leading providers of laboratory
and process technologies and equipment. Our innovative products
and high-quality services empower scientists to simplify and accelerate progress in life science and bioprocessing.
PRO Scientific is a global leader in the manufacturing of mechanical homogenizing equipment & systems for sample preparation from micro volumes to larger multi-liters. Ranging from high-quality hand-held to bench- top equipment as well as superior automated systems all made in the USA.
PRO’s unique DPS-20 and Multi-Prep Homogenizing Systems provide innovative
high-throughput homogenization solutions. PRO Generator Probes are precision crafted
offering quick, effective, and repetitive results. For over 30 years, PRO Scientific has manufactured homogenizers with unparalleled technical experience and unmatched customer support and service leading to progressive product development and expansive distribution.