microsphere integrated microfluidic disk: synergy of two techniques for rapid and ultrasensitive dengue detection
by：QY Precision 2019-08-31
Application of micro-fluid devices in diagnosis system Based on contemporary research. On the other hand, the large specific surface area of the sphere has laid an important position for its application in biometrics. Here, we report the combination of micro-spheres and micro-fluid disks in a unique hybrid platform for highly sensitive and selective detection of dengue virus. Surface Engineered Polypropylene Microspheres with well-designed functional groups facilitate biometrics in a number of ways. In order to maximize the utility of the specific surface area of the small ball in the biological molecular interaction, the micro-flow control disk is equipped with a micro-mixing system. Hybrid Mechanism ( Balloon mix) By accessing the entire sample size more effectively, increasing the number of molecular encounters between the sphere and the target analyte, resulting in signal amplification. A significant reduction in incubation time and a significant reduction in detection limits are the main motivation for the integration of microspheres in the microflow control disk. The long incubation time for routine analytical tests was reduced from 2 hours to 5 minutes, while the developed system successfully detected dengue virus in several units. The results obtained make this mixed microsphere The microfluid approach to dengue testing is a promising pathway to early detection of this deadly disease. Biological materials in the interface ( Huron, Netherlands) Through the proprietary suspension polymerization process. Different monomer including mma (MMA), 2- Ethanol (HEMA), 4-iodo-benzyloxo- Ethyl propylene ester (4-IEMA) And diester of four acetate (TEGDMA)were cross- Connected to the shape ball in the form of a polypropylene Network (Scheme 1S). Micro-presentation Before the application, in terms of storage stability, the large-scale sphere has been well characterized. As well as cell compatibility, structure and lack of soluble components. The developed micro-spheres are a bit hydrophilic in nature, which is an advantage for the intended use as a biometric platform, as they do not tend to gather in the water medium and are loosely packed in the analysis Size of polyacrylic resin Sort by screening in order to classify the sphere within the diameter range as follows: Size 1 (200–400u2009μm); size 2 (400–600u2009μm); size 3 (600–700u2009μm); and size 4 (700–900u2009μm). The sphere categories of different sizes were checked in the analytical determination, from which the diameter range was optimized (600–700u2009μm) Has been selected to integrate into a micro-fluid system (). SEM equipped with field firing gun was used to analyze the morphology of polypropylene ball in different size groups ( JSM7600F, JEOL, FESEM) Work at the acceleration voltage of 0. 5u2009kV. Samples are installed in double before Imaging To avoid surface charging, double-sided conductive carbon tape is coated with platinum. The image generated by an optical microscope calculates the size distribution of the sphere ( Olympus (Japan). Diameter measurements were performed on 500 ± 5 micron balls randomly selected in each size category. Each micro-flow control disc consists of 32 mixing units in a format of 8 kbps × Q4 4 and optimizes the geometry of the micro-flow control features based on the volume of the liquid and the maximum amount of the sphere. As shown, each mixing unit consists of an injection hole, an extraction micro-channel, and a mixing chamber with a micro-balloon. To facilitate the loading of particles into the microchamber and prevent them from escaping from the mixing device during liquid extraction, the cross-sectional area of the injection hole (4. 91u2009mm) Designed to be much larger than extracting micro-channels (0. 28u2009mm). Details of disk manufacturing and experimental setup used in the current study were previously reported. To put it simply, one is made up of three polymethyl fats (PMMA)disks ( Asia Poly industry Limited (Malaysia) A layer of latex ( Sdn flex Sdn Bhd, Malaysia) And three pressure sensitive glue (PSA)disks (FLEXcon, USA). The arrangement of plexiglass, latex and PSA layers used to make discs was demonstrated in. Mixing chamber and Micro Channel ( 700 u2009 mu m width u2009 × u2009 400 u2009 mu m depth) From the middle of the cavity and the engraved wire, the organic glass plate is digitally controlled by computer (CNC)machine ( 2525 CNC engraving and wiring, vision, USA). The CNC machine is also used to create injection/extraction holes and labels on the top of the silicone plate. Engraving Machine ( Taiwan GCC Puma II) Hired to cut Microscopic features in PSAs, as shown. The seven layers are then aligned and stacked together and placed in a screwcompressor- Clip for an hour. The micro-balloon mixing is based on the cyclic expansion and contraction of the latex film corresponding to the rotation frequency change of the disk. Centrifugal pressure at high spin rates forces the liquid to hold inside the balloon. In contrast, reducing the spin rate of the disk releases the elastic energy stored by the expanded micro-balloon and pushes the liquid back into the mixing chamber. Therefore, the frequent increase/decrease in spin rate results in the reciprocating movement of the liquid between the balloon and the mixing chamber. The change in the liquid level inside the mixing chamber corresponds to the amount of liquid that has been transferred to the balloon and is controlled by adjusting the spin rate of the disk. Details of a rotation control system customized using a high-speed camera were previously reported, which is used to optimize the rotation profile of a hybrid loop. In order to keep all Micron balls in contact with the liquid throughout the mixing process, the spin profile of the mixing cycle is determined based on the maximum weight of the Micron balls. Therefore, in the range of 600-700 μm dimensions, the mixing unit is loaded with a sphere of 80 mg and a red liquid of 100 μ l ( Select from ELISA buffer) , Similar to the volume of reagents used in routine analysis. The rotation speed is 1600 rpm, at which the liquid in the mixing chamber covers almost the micro-sphere () In the experiment, the best spin rate of the micro-flow control disk was selected. Portable spin system for ELISA experiments (and ). The micro-flow control disk is set to continuously reverse the direction of rotation after reaching the maximum speed of 1600 rpm or-1600 rpm (). During a single mixing cycle, the spin profile of the micro-fluid disk is shown in. Sandwich ELISA, known as the most specific and reliable protocol, was selected as a method of interest to minimize non- Obtain reasonable and accurate results. When the package DV ( In this case) Local fixation between two selected analytes: capture antibody (rabbit anti- Antibody to dengue virus 2, ab155042, Abcam. US) And primary antibodies ( Mouse anti DV IgG2a, ab155863, Abcam. US). The reproduction of the catch and primary antibodies occurs in different hosts, so the two biological molecules cannot bind to each other. Secondary marker antibody (anti- Alkaline ALP of mouse alkaline 2a (ab97242, Abcam US) On the contrary, the detection signal can be further merged with an anti-junction (). With the concentration of the target analyte, the signal strength of the synthesis can be amplified accordingly. The data sheet of the purchased commercial product indicates that the sandwich ELISA has the least chance of cross-cutting Reactivity or interface with other analytes (less than 2%). Pre- ELISA 96-placed microsphere with determined dose Well plates and microfluid Chambers and sandwich ELISA were performed in both. In addition, conventional ELISA was performed as a control with each group of experiments. In order to minimize experimental variability, all immunization analyses were performed using the same reagent under exactly the same conditions. All data sets obtained in this study were analyzed for standard calibration curves. No significant intraday changes were observed in the analysis performed. In order to determine the optimal dose of the sphere with the best detection performance, the sphere with different loads ( 10 mg, 20 mg, 40 mg and 80 mg) Integrated into 96- Orifice plate and micro flow control plate. Nevertheless, experiments related to the detection range, calibration curve, and comparative studies were carried out with only 20 mg of sphere ( The reasons for this choice can be further found in the text). The closed dengue virus used in this study was prepared by clinical isolation of dengue serum type 2 from patient serum samples (DV2- Isolate Malaysia M2, Gen Bank toxno. : 11062). A detailed explanation of the virus transmission and separation procedure can be found in. In addition, a detailed description of sandwich ELISA is provided. According to the purpose of the assay carried out, various viral concentrations were prepared by continuous dilution. For example, for calibration analysis, there are 4 continuously diluted viral solutions in the concentration range between 3. 5u2009×u200910u2009p. f. u/mL to 3. 5u2009×u200910u2009p. f. u/mL were used. A calibration curve is drawn by converting the data to a pair value. By running the analysis within the concentration range of 3, the detection range was investigated. 5u2009×u200910u2009p. f. u/mL to 3. 5u2009×u200910u2009p. f. u/mL. In the case of the selected virus concentration of 3, the detection signals generated from the orifice plate and the microflow control plate and the control were compared. 5u2009×u200910u2009p. f. u/mL. The negative control was calculated based on the analysis performed without a virus (nu2009=u200916). Cut- The off value for each individual system is considered to be twice the average value generated by the negative control. Only those samples that cause light density (OD) Cutting above calculation The Off value is interpreted as a positive value. All test results, and their negative controls, were drawn after subtracting the cut The value in the original data. According to the performance of the prepared Micron ball ( Get from different size categories) Conducted analysis in the orifice plate has been thoroughly evaluated and the results are given. In addition, the selected-sized sphere integrated within the micro-fluid disk was also evaluated and compared to the performance of the same-sized sphere in the orifice plate and conventional analysis. A total of 162 repetitions were detected in sandwich ELISA, including 114 positive and 48 negative samples, to study the sensitivity and specificity of the proposed method compared to traditional clinical practice ELISA. In addition, the accuracy of the analysis was calculated by true and false negative/positive readings compared to the total number of repetitions. Detection limit (LoD) Average standard deviation determined by each system ( For minimum DV concentration) Slope of calibration curve.