Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps onto a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in under 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.

We report a disposable and highly effective polymeric microfluidic viral sample concentration device capable of increasing the concentration of virus in a human nasopharyngeal specimen more than one order of magnitude in less than 30 min without the use of a centrifuge. The device is fabricated using 3D maskless xurography method using commercially available polymeric materials, which require no cleanroom operations. The disposable components can be fabricated and assembled in five minutes. The device can concentrate a few milliliters (mL) of influenza virus in solution from tissue culture or clinical nasopharyngeal swab specimens, via reduction of the fluid volume, to tens of microliters (μL). The performance of the device was evaluated by nucleic acid extraction from the concentrated samples, followed by a real-time quantitative polymerase chain reaction (qRT-PCR). The viral RNA concentration in each sample was increased on average over 10-fold for both cultured and patient specimens compared to the starting samples, with recovery efficiencies above 60% for all input concentrations. Highly concentrated samples in small fluid volumes can increase the downstream process speed of on-chip nucleic acid extraction, and result in improvements in the sensitivity of many diagnostic platforms that interrogate small sample volumes.

a) The current gold-standard of care in the management of patients with thyroid nodules is ultrasound-guided fine needle aspiration biopsy (FNAB) followed by microscopic examination of cell morphology by a trained cytopathologist. Due to the lack of distinguishing morphology, 10-25% of FNAs are termed “indeterminate” and required surgery. However, only 35% of them are found to have cancer. There is a need for a more accurate and minimally invasive cancer diagnostic technology. Our objective is to develop an inexpensive, point-of-care molecular diagnostic platform to isolate and detect thyroid specific proteins to enable real-world use of biomarkers to inform patient care. b) In this study, we engineer a miniature ion exchange column within a plastic (cyclic olefin polymer), disposable lab-on-a-chip platform for sample preparation and protein purification using microfluidic channels with a specialized porous polymer monolith (PPM)-based resin bed. We capitalized on the finding that cancer cells exhibit differential protein expressional patterns compared to normal thyroid cells by targeting thyroid transcription factor (TTF-1), a thyroid specific enhancer binding protein, as a biomarker for the diagnosis of thyroid cancer. The ability to capture and release TTF-1 from the papillary thyroid cancer (PTC) cell line was evaluated via western blot. We also tested various types of thyroid samples obtained from malignant and benign human thyroid nodules. c) We compared the efficiency of a small-scale protein prep using a commercial gravity column versus our lab-on-a-chip column. Our method showed TTF-1 protein was detectable from the lysate of 5x104 cultured BCPAP cancer cell line. In contrast, it was not detectable when purified by gravity ion-exchange column. We next showed that one can maximize the concentration of protein eluted from as fewer cells as possible. We found that by titrating the elution volume per fraction down to 10 µl, the final concentration of eluted protein can be increased, and hence increase the downstream LOD to 104 cells. Next, we evaluated our extraction and purification system by using 10 mg patient thyroid tissue samples. We tested 11 human thyroid specimens for TTF-1 protein capture. They were all found to be positive which correlated with the official histopathological findings. All tissue extracts had measurable levels of TTF-1 protein and the levels of the TTF-1 were variable in the tissue specimens. We also tested one negative thyroid specimen. No TTF-1 protein was found. d) Our microfluidic protein extraction and purification system is a platform technology that may allow for optimal use of low-volume sample preparation such as we see in thyroid biopsies. This manner, when combined with an on-chip ELISA assay, would be a simple, cost effective test that gives the doctor all the needed information quickly in a single test. When mature, the device can be applied to other type of cancer based on tailored assays for specific biomarkers. Citation Format: Shichu Huang, Siddhartha Sharma, Lena Liu, Andy Fan, Catherine Klapperich, Jennifer Rosen. Microfluidic platform for a protein-based thyroid cancer diagnostics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3494. doi:10.1158/1538-7445.AM2014-3494