The allergy diagnostic device is the point-of-care test FCP20 (DST GmbH, Germany). It is a membrane-based ELISA test for the semi-quantitative detection of allergenspecific IgE . It uses allergen extracts, reagents, and blood, serum or plasma of a patient; after 30 minutes the results of all 20 allergens can be read semi-quantitatively by the naked eye. The reagents of the current product are stored as liquids in reagent tubes and have to be cooled.
To simplify the transport and operation the test procedure had to be semi-automated. Therefore, a separate case with a flow passage and dried-up proteins within and a pump unit with a reservoir of rinsing solution are added. The advantages of this are a better transportability due to the fact that no cooling and no bottles are needed, as well as a less time consuming procedure for the user. The additional pump unit controls the whole process and ensures that the results of the analysis are independent from the user. The whole system will consist of three components; two disposable cartridges (detector and protein resolve system) and one stationary (pump unit and reservoir). Figure 1 shows the described devices in a schematic diagram.
Subject of the present paper is the establishment of the LIF-method, which is used to describe the release kinetics of surface-bound proteins . In order to describe the resolve process of dried-up proteins in fluid (e.g. NaCl 0.9%) different analyses were made. First of all, the concept had to be proven. In other words, the possibility to rediscover the resolved quantity of protein in relation to the brought in had to be demonstrated. Secondly the dye-protein combination which works best had to be found and the lowest concentration, at which the microscope-LIF-system works, had to be determined. Thirdly the ideal volume and concentration of dried-up proteins which is needed to transport the dissolved proteins to the scope had to be determined experimentally. Finally, it was important to observe whether the resolved protein in the volume flow was homogenous, granular, in strings or something different.
The analyses were made with an optical study method named laser-induced fluorescence (LIF) . The system detects if dyed labelled proteins emit light when illuminated with light of a specific wavelength. The LIF-system is combined with a stereo-microscope and observes and measures the processes in the microfluidic protein storage and resolve device.
The investigations are divided into three stages. First of all, a calibration for the concentration is performed in order to assign the measured light intensities to a concentration. After this, the drying and resolving processes of a drop of dye-protein solution are studied. The investigations are then completed by measuring the volume flow of the resolved protein streaming towards the detector.
The stereo-microscope Discovery V20 is manufactured by Carl Zeiss (Göttingen, Germany), the LIF-system and the imaging application software were provided by LaVision (Göttingen, Germany). The aperture is wide open. The light source is a mercury lamp operated at full intensity. Zoom is 46x with a zoom lens of 10x. The filter set has the number 43 Cy3/Rhod/RFP (D) from Carl Zeiss (EX BP 545/25, BS FT 565, EM BP 606/70). The camera system “Imager sCMOS” has a sCMOS-chip and a resolution of 2560x2160 pixels. Images are made in single-frame mode with an exposure time of 100 ms. The resolving fluid is NaCl 0.9%.
Every displayed figure is characterised by its colour resolution or, more specifically, its range of counts, where the lowest measured concentrations are blue or nearly black and the highest concentrations are displayed in red. Figures which belong together have the same colour resolution to ensure comparability (e.g. 0 – 25.000 counts).
2.1 Calibration process
A great number of dye-protein combinations are available on the market. On the one hand, a qualified combination has to meet the requirements of the POCT allergy diagnostic and be a standard of comparison for the proposed analyses. On the other hand, the LIF-system has its own settings and requirements (e.g. filters, beam splitter, light source and emission spectrum).
AlexaFluor 532 and 546 from Life Technologies (Darmstadt, Germany) fulfil both the mentioned requirements. Both combinations are compared by means of the calibration process and their intensity of luminescence. This intensity depends linearly on the concentration of the dye-solvent-mix. A greater intensity leads to a more sensitive detection of lower concentrations.
Within the calibration process, different known concentrations are observed and processed in order to obtain a valid calibration for later testing. The measured light intensity in later analyses will then be processed and calculated in the same way and compared with the calibration. This way, the measured light emission results in a value for the concentration
2.2 Drying and resolve process
At this point the release kinetics is observed in a static way. In other words, a known amount of concentration of a protein-dye solution is dried-up in a flow passage. Then the dry-process is observed during the following hours. After this, the same volume (10 µl) of a NaCl-solution is added. The resolving process is observed and is displayed in a time lapse of about half an hour.
The investigations are executed without any volume flow. Only capillary forces and the resolving process lead to any observed mixing.
2.2 Flow process
The resolve process is examined under known conditions, i.e. for a given dye-protein combination and known values of the concentration, volume of the dried-up solution and the volume flow. The fluid (NaCl 0.9%) resolves the thin film of dye-protein and takes an unknown amount of concentration with it.
The following figure presents an example of the analysed flow passages. The channel has a length of 6 cm, a width of 2 mm and a depth of 0.3 mm. The cover and the channel are both made of polycarbonate. The focus of the microscope is in the centre of the figure during the observation of the flow.
3.1 Calibration process
At this point, the dye-protein combinations (AF532 and AF546) are analysed concerning their suitability for further testing. The results are shown in the following figures. To ensure comparability, the figures have the same colour resolution (0 – 25.000 counts). As previously mentioned, lower concentrations are blue or dark, whereas the highest concentration is shown in red.
Comparing Figures 3 and 4 reveals that AF546 has a higher light intensity. The absorption and emission fit better than for AF532. The following dry and resolve observation are made with AF532. The flow process is observed with AF546.
Figure 5 shows the relationship between the sample concentration and the measured light intensity. A mixture of 0.01 mg dye-protein per millilitre stands for a concentration of 1 %, a mixture of 1.00 mg per ml for 100%.
3.2 Drying and resolve process
Figure 6 illustrates the drying process of a 0.5 mg/ml dye-protein-solvent solution. The time lapse shows a decrease in light intensity. The initial concentration of about 50% decreases to at least 2% due to the drying process. The volume of the homogenous solution from the left picture evaporates. Parts of the fluorescent stick to the edge of the flow passage or cake at the bottom (right picture). The volume of 10 µl has disappeared and the protein-dye solution has become a thin film.
Figure 7 shows the resolving process. Little grainy fragments appear immediately and flow to the centre due to capillary forces. A homogenous solution appears within 30 minutes and the concentration increases to at least 6%.
3.3 Flow process
The green, red and white lit area in Figure 8 is a dried-up drop of dye-protein solution. The little black and dark-blue point in its centre is a marker, made with a pencil and pointed onto the transparent cover of the channel.
The first image in Figure 9 displays the frontline of the flow field and a volume of circa 1µl. The concentration is nearly 10% and seems uniform except for some strings in the centre of the flow. The second image, which is recorded 3 seconds later, shows a concentration which is at least 4 times higher. In the third image, recorded 6 seconds after the flow started and protein began to resolve, the concentration is increased by at least 15% and seems to distribute itself homogeneously. The last image, taken 12 seconds after the delivery started, shows a decrease in concentration and a more and more grainy solution.
The second time lapse in Figure 10 shows another flow passage. The conditions are the same as above and the pictures are recorded at the same time steps.
Unlike the process in Figure 9, this resolving process is non-uniform and less homogeneous. In the first image the concentration is about 7% and shows strings and fragments. The concentration in the middle of the flow in the second image is approximately 18% and contains grains as well. The third image shows even more particles and grainy fragments. Finally, after 12 seconds of streaming, the concentration decreases to at least 7% with several highlighted fragments.
The resolving occurs within the first second of wetting in all analysed flow passages. Within a few seconds, the concentration rises to a relatively high level and decreases afterwards. However, the flow has no constant level of concentration and appears in strings or grainy fragments in certain cases. Although, the initial conditions were identical, the maximum level of concentration in one of the considered cases is only the half of the other one.
The method of measuring the drying and resolving process with the use of a stereo-microscope combined with the LIF-system works accurately and provides satisfying results, as expected. Whether in a static or dynamic way, the concentration of the observed processes can be measured precisely and visualized in high resolution. Low concentration levels and how the dye-protein solution is resolved in the flow (e.g. homogenously, granular, in strings or something else) can be measured and observed.
Further studies have to be performed in order to get a better understanding of the resolving process and the mixing of the dye-protein in the flow . The high deviation of the peak-concentration and the time curve of the concentration will be another aspect of interest. Its cause is currently suspected in the dispensing system. When the solvent dye-protein solution is brought onto the flow passage, the fluid volume is forced by capillary forces and the dried-up drop forms a more or less random formed thin-film (see Figure 8). These thin-films have different thicknesses, areas and form.
Funding: This project is funded by the Bundesministerium für Bildung und Forschung (FKZ: 03WKCC 10; Innovative Regional Growth Cores “Centifluidic Technologies”) and is part of the BMBF-initiative “Entrepreneurial Regions” “The BMBF Innovation Initiative for the New German Länder“.
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