**1.3 Approaches and technical issues**

Establishment of biometrology is technically demanding. Biological materials are mostly large and complex in their chemical structure. Typical dimensions of important biological entities are illustrated in Fig. 1.1. Even with a highly advanced analytical technique such as mass spectrometry, biological materials are not readily quantifiable with a desired level of accuracy. There are substantial technical gaps between the demands and currently available techniques.

Fig. 1.1. Dimensions (Stokes-Einstein diameters) of biologically important materials. The dimensions of nucleic acids is indicated in length.

Then, approaches to overcome such technical gaps need to be sought out. The approaches suggested by Korea Research Institute of Standards and Science (KRISS, www.kriss.re.kr) scientists as basic approaches for establishment of metrology for biology (Fig. 1.2) would not be much disputable. The first coming idea is to reduce large biological molecules to smaller chemical units that can be accurately quantifiable with current analytical techniques. Reduction to chemical units can be achieved through chemical reactions or enzymatic reactions. The quantity of the original biological molecules is then calculated from the knowledge on their chemical compositions. Uncertainty of such analytical procedures mainly arises from the degrees of perfection of such reduction processes. As far as metrological assessment is made on the uncertainty of the reduction process, the overall analytical procedure can be regarded as of metrological quality. Quantitation of proteins by amino acid composition is a typical example of the reduction approach. Examples of this approach including metrology for protein quantification are described in detail in the following sections.

Large biological materials are discrete and countable. Quantification of cells (cytometry) is an excellent example of count-based quantitation of which history is now longer than several decades. We can extend this approach whatever biological materials that have a discrete (countable) nature in principle. Quantification of large DNA particles is described in the following section as well as digital-polymerase chain reaction (digital-PCR) that can be also categorized into count-based quantification. In quantification of microorganisms, counting of cultured colonies is of a relatively long history.

Fig. 1.2. Approaches for establishment of technical basis for metrology for biology suggested by KRISS scientists (from the presentation at 2005 CCQM workshop on "New Challenges for the development of Primary or Higher Order Measurement methods and Procedures").

Several important biological quantities such as activity, viability, efficacy, and toxicity are not the quantities of biological molecules as above. Instead, these quantities are defined by the experts group of the related fields. These quantities are often called "method-dependent quantities" as those are determined by following the analytical procedures agreed among the experts groups. Metrology for such quantities is therefore of complexity and is often thought to be out of the domain of metrology. However, comparability of the measurement of those quantities is of great importance, and a great deal of improvement can be made by applying metrological rigorousness to the physical or chemical conditions involved in each analytical procedure. An example of such aspect is demonstrated in PCR in the following section. Noticeable technical advances have been made in metrology for quantification of nucleic acids and also some for proteins, but not yet for other complex biological materials such as cells and glycans. For these materials, a great deal of research should be done to visualize the practical paths forward realization of metrology. Here, only currently conceivable technical issues are introduced.
