Prediction of acute rejection in kidney transplanted patients based on the point-of-care isothermal molecular diagnostics platform.

In this study, we proposed an advanced point-of-care molecular diagnostic technology to evaluate the acute rejection (AR) in kidney transplanted patients. On the contrary to the conventional PCR method, we developed a colorimetric loop mediated isothermal amplification (LAMP) for quantitative analysis of the six biomarkers related to AR (CD3ϵ, IP-10, Tim-3-HAVCR2, CXCL9, PSMB9, C1QB) with a reference gene (18S rRNA). Using urinary cDNA samples of transplanted patients, it turned out that three biomarkers among six, namely IP-10, Tim-3-HAVCR2 and C1QB, have significant discrepancy in quantity between the stable graft (STA) patient and the AR patient. The AR prediction model using these three biomarkers was established, which could estimate the immune-rejection in the patients with 93.3% of accuracy. For the point-of-care (POC) molecular diagnostics for the AR evaluation, we constructed a centrifugal microfluidic platform, in which the RNA extraction from the clinical urinary samples, the quantitative reverse-transcription (RT)-LAMP reaction, and the data analysis based on the AR prediction model could be performed in a serial order. Ten blind clinical samples were analyzed on the POC genetic analyzer, showing 100% match with the validated qPCR data. Thus, the proposed advanced molecular diagnostic platform enables us to perform the timely treatment for the transplanted patients who are suffering from the allograft failure and side effects such as infection and malignancy.

High-throughput human DNA purification on a centrifugal microfluidic device for rapid forensic sex-typing.

We introduce a novel centrifugal disc for purifying nucleic acid (NA) in a high-throughput manner to perform the human sex-typing of forensic samples. The centrifugal disc was designed with double-sided etched channels to fabricate 30 extraction units, which was capable of purifying 30 forensic samples in a single run. In order to introduce the washing solution (70% ethanol) and the elution buffer for the 30 extraction units in an automatic manner, we designed the aliquoting chambers that were connected with a zigzag delivery channel. The super-hydrophobic zigzag-shaped aliquot structure plays a crucial role in automatically dividing the washing solution and the elution buffer into 30 aliquots with one injection shot. The Whatman glass filter paper was used as an NA extraction matrix and sophisticated passive valves were equipped to avoid the overflowing of these buffers to the neighboring chamber during the injection. To operate the disc, we developed a portable workstation that consists of a buffer storage system, a buffer injection system, and a spinning unit. The entire process was automatically operated by the in-house portable workstation. Genomic DNA extraction using thirty forensic samples was completed in 10 min. Using the purified genomic DNA, we performed a loop-mediated isothermal amplification (LAMP) reaction for sex-typing by targeting the human alphoid repeat sequence of the Y-chromosome and the human 18S rRNA. The combination of the high-throughput centrifugal disc for NA extraction and the LAMP reaction enables us to complete the genetic sex-typing in 30 min.

Quantification of colorimetric isothermal amplification on the smartphone and its open-source app for point-of-care pathogen detection.

The increasing risk of infectious pathogens, especially in the under-developed countries, is demanding the development of point-of-care (POC) nucleic acid testing in the low-resource setting conditions. Here, we describe a methodology for colorimetric quantitative analysis of nucleic acid using an easy-to-build smartphone-based platform, offering low-cost, portability, simplicity in operation, and user-friendliness. The whole system consists of a hand-held box equipped with a smartphone, a film heater, a white LED, a loop-mediated isothermal amplification (LAMP) chip, and a DC converter, and all the processes were powered by a portable battery of 5 V. Upon the amplification of the target gene by an Eriochrome Black T-mediated LAMP reaction, the color of the LAMP reaction was changed from violet to blue that was real-time recorded by a smartphone camera. To keep track of the progress of the color change, we developed a novel mobile app in which a hue value was accepted as an indicator for color transition and for determining the threshold time of the amplification reaction. A calibration curve could be generated by plotting the logarithm of the known concentration of the DNA templates versus the threshold time, and it can be used to predict the copy number of nucleic acids in the test samples. Thus, the proposed mobile platform can inform us of not only qualitative but also quantitative results of the pathogens. We believe that this advanced colorimetric approach and the mobile app can expand the potentials of the smartphone for the future POCT system in the bio-diagnostic fields.