High-throughput screening

High-throughput screening (HTS) is a method for scientific experimentation especially used in drug discovery and relevant to the fields of biology and chemistry. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, High-Throughput Screening or HTS allows a researcher to quickly conduct millions of biochemical, genetic or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies or genes which modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.

Methodology and equipment

In essence, HTS uses automation to run an assay, or screen, of a library of candidate compounds against a target. An assay is a test for specific activity: usually inhibition or stimulation of a biochemical or biological mechanism. Typical HTS screening libraries or "decks" can contain from 100,000 to more than 2,000,000 compounds (circa 2008).

Assay plate preparation

The key labware or testing vessel of HTS is the microtiter plate: a small container, usually disposable and made of plastic, that features a grid of small, open divots called wells. Modern (circa 2008) microplates for HTS generally have either 384, 1536, or 3456 wells. These are all multiples of 96, reflecting the original 96 well microplate with 8 x 12 9mm spaced wells. Most of the wells contain experimentally useful matter, often an aqueous solution of dimethyl sulfoxide (DMSO) and some other chemical compound, the latter of which is different for each well across the plate. (The other wells may be empty, intended for use as optional experimental controls.)

A screening facility typically holds a library of stock plates, whose contents are carefully cataloged, and each of which may have been created by the lab or obtained from a commercial source. These stock plates themselves are not directly used in experiments; instead, separate assay plates are created as needed. An assay plate is simply a copy of a stock plate, created by pipetteing a small amount of liquid (often measured in nanoliters) from the wells of a stock plate to the corresponding wells of a completely empty plate.

Reaction observation

To prepare for an assay, the researcher fills each well of the plate with some biological entity that he or she wishes to conduct the experiment upon, such as a protein, some cells, or an animal embryo. After some incubation time has passed to allow the biological matter to absorb, bind to, or otherwise react (or fail to react) with the compounds in the wells, measurements are taken across all the plate's wells, either manually or by a machine. Manual measurements are often necessary when the researcher is using microscopy to (for example) seek changes or defects in embryonic development caused by the wells' compounds, looking for effects that a computer could not easily determine by itself. Otherwise, a specialized automated analysis machine can run a number of experiments on the wells (such as shining polarized light on them and measuring reflectivity, which can be an indication of protein binding). In this case, the machine outputs the result of each experiment as a grid of numeric values, with each number mapping to the value obtained from a single well. A high-capacity analysis machine can measure dozens of plates in the space of a few minutes like this, generating thousands of experimental datapoints very quickly.

Depending on the results of this first assay, the researcher can perform follow up assays within the same screen by "cherrypicking" liquid from the source wells that gave interesting results (known as "hits") into new assay plates, and then re-running the experiment to collect further data on this narrowed set, confirming and refining observations.

Automation systems

Automation is an important element in HTS's usefulness. Typically, an integrated robot system consisting of one or more robots transports assay-microplates from station to station for sample and reagent addition, mixing, incubation, and finally readout or detection. An HTS system can usually prepare, incubate, and analyze many plates simultaneously, further speeding the data-collection process. HTS robots currently exist which can test up to 100,000 compounds per day. The term uHTS or ultra high throughput screening refers (circa 2008) to screening in excess of 100,000 compounds per day.

Techniques for increased throughput and efficiency

Unique distributions of compounds across one or many plates can be employed to increase either the number of assays per plate, or to reduce the variance of assay results, or both. The simplifying assumption made in this approach is that any N compounds in the same well will not typically interact with each other, or the assay target, in a manner that fundamentally changes the ability of the assay to detect true hits.

For example, imagine a plate where compound A is in wells 1-2-3, compound B is in wells 2-3-4, and compound C is in wells 3-4-5. In an assay of this plate against a given target, a hit in wells 2, 3, and 4 would indicate that compound B is the most likely agent, while also providing three measurements of compound B's efficacy against the specified target. Commercial applications of this approach involve combinations in which no two compounds ever share more than one well, to reduce the (second-order) possibility of interference between pairs of compounds being screened.

Recent advances

In March 2010 research was published demonstrating an HTS process allowing 1,000 times faster screening (100 million reactions in 10 hours) at 1 millionth the cost (using 10^-7 times the reagent volume) than conventional techniques using drop-based microfluidics. Drops of fluid separated by oil replace microplate wells and allow analysis and hit sorting while reagents are flowing through channels.

In 2010 researchers developed a silicon sheet of lenses that can be placed over microfluidic arrays to allow the fluorescence measurement of 64 different output channels simultaneously with a single camera. This process can analyze 200,000 drops per second.

Increasing lab utilization of HTS

HTS is a relatively recent innovation, made lately feasible through modern advances in robotics and high-speed computer technology. It still takes a highly specialized and expensive screening lab to run an HTS operation, so in many cases a small-to-moderately sized research institution will use the services of an existing HTS facility rather than set up one for itself.

There is a trend in academia to be their own drug discovery enterprise. ( High-throughput screening goes to school) Facilities which normally only industry had can now increasingly be found as well at universities. UCLA for example, features an HTS laboratory (Molecular Screening Shared Resources (MSSR, UCLA) which can screen more than 100,000 compounds a day on a routine basis. The University of Illinois also has a facility for HTS, as does the University of Minnesota. The Rockefeller University, has an open access (infrastructure) HTS Resource Center HTSRC (The Rockefeller University, HTSRC) which offers a library of over 165,000 compounds.

In the United States, the National Institute of Health or NIH has created a nationwide consortium of small molecule screening centers that has been recently funded to produce innovative chemical tools for use in biological research. The Molecular Libraries Screening Center Network or MLSCN performs HTS on assays provided by the research community, against a large library of small molecules maintained in a central molecule repository.([2])

For more information see Laboratory automation

See also

• Drug discovery hit to lead
• Virtual high throughput screening
• High-content screening
• Drug discovery
• Z-factor
• Compound management
• Synthetic genetic array
• Yeast two-hybrid screening

Further reading

• Staff (2008-08-01). "High-Throughput Screening Challenges". Genetic Engineering & Biotechnology News. Drug Discovery Rountable Discussion. Vol. 28, no. 14. Mary Ann Liebert. pp. 26–27. ISSN 1935-472X. Retrieved 2008-10-01.{{cite news}}: CS1 maint: date and year (link)

External links

• Molecular Screening Shared Resources (MSSR, UCLA)
• Advanced Cell Classifier (ACC) project for high-throughput screen evaluation (ETH, Zurich)
• Yale Center for High Throughput Cell Biology
• Biohts: HTS resources and links- Biohts
• Society for Biomolecular Sciences - links (SBS)
• Open Screening Environment
• High-throughput screening technology
• Setting up High-Throughput Screening Laboratory (Koppal, Lab Manager Magazine)
• Assay Guidance Manual (NIH, NCGC)