An enzymatic analyzer is complex equipment, but at the same time very simple to use and capable of greatly facilitating laboratory work. Essentially, it is a robot that repeatedly performs a series of steps in carrying out the analysis: taking the sample and reagents, mixing and measuring the changes in the optical density of the specific reaction mixture for each parameter analyzed. The internal algorithms then process the readings and return a concentration value of the analyte in the sample. And so on over and over again until we complete the requested test list. Easy.
But under this apparent simplicity a multitude of critical details are hidden so that the measured result is the one that really reflects the characteristics of the sample. In this post we will comment on some of them incorporated into our DIONYSOS systems.
A critical point is that of sampling, especially with regard to precision (i.e., the capacity to consistently deliver exactly the same amount of sample), especially if we take into account that we are talking about quantities that usually range from 3 to 10 uL (as a reference, it is considered that the size of a raindrop would be about 50 uL). The typical resolution of 0.1 uL would result in a maximum random variation of 0.05 uL, or what is the same, a CV% associated with the dispensing of a 3 uL sample of a maximum of 1.7%. This is achieved through the use of injection pumps with ceramic pistons capable of working up to 5 million cycles without deformation.
But in addition to being precise, that same tip has to avoid contamination between consecutive samples, between samples and reagents, and between different reagents (also called carry-over). Since the tip in an analyzer is not a disposable item, the surface treatment of the tip, both internal and external, is the determining technological factor. In the past, Teflon-treated tips were used, although they have been displaced by modern nanomaterials and, as in the case of our DIONYSOS analyzers, in which much more efficient ultra-hydrophobic surfaces are achieved.
After the sample and reagents have been deposited in the reaction cell, a stirrer homogenizes the mixture to ensure that the reaction proceeds smoothly, without unexpected concentration gradients or trapped air bubbles on the cell walls. In this way a stable and monotonic signal is achieved.
Temperature is an element that intervenes in two different facets. On the one hand, enzymatic reagents require a low conservation temperature that lengthens their useful life and maintains their stability; on the other, enzymes need a specific temperature to function and, to achieve a stable reaction, constant throughout the reaction and the same in all cells.
An efficient cooling system keeps reagents in optimum condition, even when the analyzer is not running. When reagents are aspirated, they should be quickly heated inside the tip to bring them closer to operating temperature but without damaging them. Once dispensed into the cuvette, the first cycles are essential to reach the reaction temperature of the mixture thanks to a heating system by hot air circulating between the reaction cells. As cells allows some space between them, this air flow both maintains the temperature and prevents a reaction from cooling down when reagent is added to the adjacent cell.
The ability of a substance to interact with a specific wavelength is called absorbance, which is nothing more than the difference between the light that reaches a solution containing that substance and that which comes out after passing through it. This magnitude is proportional to the concentration of said substance in the medium (the so-called Beer-Lambert Law). When that substance is transformed as a consequence of a reaction, the absorbance of the sample changes and therefore we can calculate how much its concentration has changed. Most techniques monitor the absorbance change of the coenzyme NAD + when it is transformed into NADH (which absorbs light at 340 nm); but other techniques will use different wavelengths. The selection of the suitable wave is carried out by means of specific filters that allow a very narrow bandwidth (+/- 2 nm), together with highly sensitive photodiodes capable of registering signals of up to one ten thousandth absorbance.
To have a stable reading, often the first absorbance measurement is made before adding the second reagent (or the sample, in case of monoreactive reactions) and the second absorbance measurement when the reaction has completely finished. A particular characteristic of the DIONYSOS analyzers is that the user can configure the number of measurements to be carried out both at the beginning and at the end of the reaction, so that the random errors associated with the measurement itself can be compensated, which significantly improves the precision of the same. In addition, there are other factors that produce oscillations in the signal that should be accounted an compensated, such as the drift of the lamp temperature itself or the presence of dust in the optical path, so it is achieved through incorporating a cooling system for the lamp and sealing the optical system as a whole so that it is not be affected by the environmental conditions.
All these systems work in a coordinated and efficient way so that the analyst only has to deal with feeding the work routines. And even this task can be simplified with the help of other optional support elements, such as integration within a laboratory management system, identification of samples by barcode or the design of an intuitive and visual navigation interface.
The DIONYSOS system is a state-of-the-art analyzer designed to do daily work in a simple and efficient way, freeing the technician from the most repetitive and error-prone tasks.
Enzyme systems and FT-NIR are totally different systems and a direct comparison between them is not possible, except when we refer to specific process needs. Most often, the two systems are used together, amplifying the advantages and eliminating the disadvantages. In many warehouses, during periods of intense work, such as harvesting, FT-NIRs are used to process numerous samples in a short time, obtaining sufficient initial results for this purpose. Later, however, the use of enzyme systems allows the results to be verified with greater precision and accuracy and a greater guarantee of data security. Additionally, enzyme systems can be used as a simple, inexpensive, and safe method to verify and calibrate FT-NIR systems.
For more than 10 years, Sinatech’s commitment to the winemaker has been working side by side to provide the most appropriate analytical solutions to the control and monitoring of the winemaking process. Automated methods easily adaptable to any work routine, with a personalized advisory team to help you quickly and smoothly implement.