Calcium ions are important for muscle contraction, nerve impulse transmission, bone mineralization, and many other purposes. As is the case for many metal ions, enzymes, hormones, and other biologically relevant species, the amount of calcium ions free in the blood is tightly regulated.
A large deviation in blood calcium levels is typically indicative of, or will cause, a disease, such as cancer. Thus, it is important to have an easy, cheap, quick method to quantitate the amount of calcium in a blood sample.
Sangyong Jon (Gwangju Institute of Science and Technology, South Korea) and coworkers have developed such an assay. Their protein-coated gold nanoparticles exhibit a color change when they bind to calcium ions, enabling simple and rapid diagnostics.
Basis of the assay.
The general concept utilized by these scientists is not new. Many research groups in the past ten years or so have developed variants of this assay for analytical detection purposes.
The assay is based on the fact that the color of a water-based solution of gold nanoparticles depends on the diameter of the nanoparticles. By developing a protocol whereby the analyte of interest aggregates the nanoparticles (thus changing their diameter), a color change is observable, indicative of the analyte being present, and also enables determination of the concentration of the analyte.
These scientists have used calsequestrin proteins as the molecules that enable nanoparticle aggregation. Calsequestrin is a calcium-binding protein, found in certain types of muscle, that has the function of storing and releasing calcium ions.
In the absence of calcium ions, calsequestrin proteins are unfolded. However, when they bind calcium ions, the protein becomes more compact, then binds to a neighboring protein, and eventually polymerizes.
Thus, gold nanoparticles coated with calsequestrin proteins will form aggregates, effectively changing the diameter of the gold nanoparticles. This results in an easily observable color change.
There are a number of conditions that this type of assay should meet if it is to be practically useful. Nanoparticle aggregation should be reversible, the assay must be selective and sensitive, and the assay must work in blood serum; these are what the scientists investigated next.
Nanoparticle aggregation is reversible.
The nanoparticles should aggregate reversibly. This would enable the assay to be performed with the same batch of nanoparticles multiple times, reducing the cost of the assay.
The scientists added EDTA, ethylenediaminetetraacetic acid, to a solution of aggregated protein-coated nanoparticles. EDTA strongly binds to calcium ions, much more strongly than calsequestrin.
After calcium ion binding to EDTA, nanoparticle aggregation reversed, presumably because the protein polymerization reversed. Reversible aggregation was observed through up to three aggregation cycles.
Calcium ion detection is selective.
There are many other small metal ions present in blood serum, in addition to calcium ions. These ions must not interfere in the scientists' assay.
The scientists tested their protein-coated nanoparticles against many other positively charged metal ions, besides calcium, Among them were magnesium, sodium, and zinc ions, all of them at far higher concentrations than that found in blood.
None of them interferred in the assay. This is especially noteworthy, because most assays for either calcium or magnesium ions are not selective for one over the other.
Calcium ion detection is sensitive.
The scientists needed to demonstrate that their assay is selective for biologically-relevant calcium ion concentrations, under biologically-relevant conditions. This problem has plagued other color-based calcium detection schemes.
The scientists found that the amount of color change (nanoparticle aggregation) is linear with respect to calcium ion concentration. The linear concentration range is from 40 to 160 parts per million calcium ions.
This well encompasses the 96 parts per million healthy concentration that is normally observed. It also encompasses the up to 140 parts per million concentration observed in certain cancers.
The assay is effective in blood serum.
In order for a blood serum-based medical assay to be practically useful, it must work in real-world samples, not just under laboratory conditions. The scientists tested their assay under these more rigorous conditions.
Preliminary results gave an observable color change in response to calcium in blood serum. Thus, this protocol is not just a "lab toy."
Overall evaluation.
These scientists have developed an analytical protocol for quantifying calcium ion concentration in blood serum, based on an easily observable color change, and it requires no (or common) instrumentation. The response is linear, sensitive, selective, and reversible, all features of a practically useful assay that should find its way into medical settings.
for more information:
Kim, S., Park, J. W., Kim, D., Kim, D., Lee, I.-H., & Jon, S. (2009). Bioinspired Colorimetric Detection of Calcium(II) Ions in Serum Using Calsequestrin-Functionalized Gold Nanoparticles Angewandte Chemie International Edition, 48 (23), 4138-4141 DOI: 10.1002/anie.200900071