Background: Detection or monitoring of brain damage is a clinically crucial issue. Nucleic acids in the whole blood can be used as biomarkers for brain injury. Polymerase chain reaction (PCR) which is one of the most commonly used molecular diagnostic assays requires isolated nucleic acids to initiate amplification. Currently used nucleic acid isolation procedures are complicated and require laboratory equipments. Objective: In this study, we tried to develop a simple and convenient method to isolate nucleic acids from the whole blood sample using a tiny battery-powered electric device. The quality of the isolated nucleic acids should be suitable for PCR assay without extra preparation. Methods: A plastic device with separation chamber was designed and printed with a 3D printer. Two platinum electrodes were placed on both sides and a battery was used to supply the electricity. To choose the optimal nucleic acid isolation condition, diverse lysis buffers and separation buffers were evaluated, and the duration and voltage of the electricity were tested. Western blot analysis and PCR assay were used to determine the quality of the separated nucleic acids. Results: 2ul of whole blood was applied to the cathode side of the separation chamber containing 78 ul of normal saline. When the electricity at 5 V was applied for 5 min, nucleic acids were separated from segment 1 to 3 of the separation chamber. The concentration of nucleic acids peaked around 7~8 mm from cathode side. PCR assay using the separation buffer as the template was performed successfully both in conventional and realtime PCR methods. The hemoglobin in the whole blood did not show the inhibitory effect in our separation system and it may be due to structural modification of hemoglobin during electric separation. Conclusion: Our simple electric device can separate nucleic acids from the whole blood sample by applying electricity at 5 V for 5 min. The separation buffer solution taken from the device can be used for PCR assay successfully.
Background: Stroke is one of the leading causes of death and disability in adulthood worldwide. A simple and convenient diagnostic method is needed for monitoring high-risk patients for stroke. Few POCTs are available for stroke diagnosis. Soluble blood P-selectin is known as a biomarker for platelet aggregation. Increased expression of P-selectin is observed in coronary artery disease, acute myocardial infarction, stroke and peripheral arterial disease. Objective: A simple method that can measure the increased expression of P-selectin in stroke patients is intended to be used for diagnosis or early detection and hospital monitoring of ischemic stroke. Method: Plasma proteins in blood were separated using a three-layered filter system. Quantum dot and antibody were conjugated to detect biomarkers present in plasma and then measured with a fluorescence spectrophotometer. Results: The detection limit of soluble P-selectin confirmed by immunoassay was 1 ng/ul. In order to increase the sensitivity and simplify the reaction, the detection limit was measured to evaluate the sensitivity of the quantum dot labeled anti P-selectin antibody. As a result, P-selectin of 5 ng/ul or more showed saturation signal intensity, indicating the upper limit of detection, and 10 pg/ul was the lower limit of detection. Conclusion: In this study, we proposed a three-layer filter membrane system that can separate biomarker-rich fractions from whole blood, simplifying the analysis process and improving sensitivity by using quantum dot-labeled antibodies to detect biomarkers. We hope that our system complements the advantages of POCT and can be applied to real clinical applications.
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