Proteins on the cell surface are involved in cell-cell contact, shuttling other molecules into the cell, and many other important functions. They are also often used to diagnose a disease.
For example, if there is a specific protein on the cell surface, or an abnormal amount of it, this can be indicative of cancer. It doesn't have to be cancer; it could be any one of many other conditions, depending on the protein.
Cell sorting instrumentation is commonly used to detect these cell-surface proteins. However, such instrumentation is usually insufficient for detecting proteins that are present in low numbers.
Sometimes there may only ever be small numbers of a specific protein on the cell surface. Alternatively, there may be small numbers early on in a disease.
It is easy to envision how detecting the previously undetectable would be of medical benefit, and enable scientists to carry out investigations that had previously been inconceivable. Consequently, a protocol that enables the detection of small numbers of proteins on the cell surface would be useful.
Haakan Joensson (Royal Institute of Technology, Sweden) and coworkers have used a microfluidic device to tackle this challenge. Their device can be used to detect small numbers of protein molecules, while simultaneously sorting the desired cells from the others for purification and subsequent analysis.
The protocol.
The scientists' detection protocol first "amplifies" the cell-surface protein. Briefly, many fluorescent molecules are produced for every cell-surface protein, effectively turning one molecule to be detected into many molecules.
The scientists chemically bound the enzyme β-galactosidase to the cell-surface proteins of their cell sample. This enzyme, when heated to 37°C, turns on fluorescence from molecules in the microfluidic device.
Thus, in a time- and protein concentration-dependent manner, many fluorescent molecules are produced for every one of the cell-surface proteins present (the ones that the scientists are trying to detect). This is how amplification is achieved.
Detection and sorting.
A major purpose of this research was to use this standard amplification protocol in their device, to enable sensitive cell-surface protein detection and cell sorting. What is the detection limit using their device?
The scientists found that they could detect as few as 22 protein molecules per cell. Additionally, they could adjust the experimental conditions such that one cell was isolated into one 40 micron (40 millionths of 1 meter) diameter drop in their device.
This enables one positively identified cell to be sorted from all the rest, at a rate of 2500 drops (cells) per second. The scientists successfully sorted a batch of cells possessing the cell-surface protein CCR5 (chemokine receptor 5), which is a low-abundance protein that HIV utilizes to enter cells.
Ninety-five percent of the CCR5 cells could be discriminated from background cells. This is much improved over the 22% achieved with conventional cell sorting instrumentation, with similar cell sorting speed.
Future prospects.
These scientists have used a standard amplification protocol to detect small numbers of cell-surface proteins, and sort the cells possessing these proteins for subsequent analysis. It is simple and much improved over conventional cell sorting instrumentation, which should make it a valuable tool for biomedical purposes.
for more information:
Joensson, H. N.; Samuels, M. L.; Brouzes, E. R.; Medkova, M.;
Uhlén, M.; Link, D. R.; Andersson-Svahn, H.
Detection and analysis of low-abundance cell-surface biomarkers
using enzymatic amplification in microfluidic droplets.
Angew. Chem. Int. Ed. 2009, 48, 2518-2521.