A Novel On‐Chip Method for Differential Extraction of Sperm in Forensic Cases

One out of every six American women has been the victim of a sexual assault in their lifetime. However, the DNA casework backlog continues to increase outpacing the nation\u27s capacity since DNA evidence processing in sexual assault casework remains a bottleneck due to laborious and time‐consuming differential extraction of victim\u27s and perpetrator\u27s cells. Additionally, a significant amount (60–90%) of male DNA evidence may be lost with existing procedures. Here, a microfluidic method is developed that selectively captures sperm using a unique oligosaccharide sequence (Sialyl‐LewisX), a major carbohydrate ligand for sperm‐egg binding. This method is validated with forensic mock samples dating back to 2003, resulting in 70–92% sperm capture efficiency and a 60–92% reduction in epithelial fraction. Captured sperm are then lysed on‐chip and sperm DNA is isolated. This method reduces assay‐time from 8 h to 80 min, providing an inexpensive alternative to current differential extraction techniques, accelerating identification of suspects and advancing public safety

Point-of-care vertical flow allergen microarray assay: proof of concept

BACKGROUND:\ud Sophisticated equipment, lengthy protocols, and skilled operators are required to perform protein microarray-based affinity assays. Consequently, novel tools are needed to bring biomarkers and biomarker panels into clinical use in different settings. Here, we describe a novel paper-based vertical flow microarray (VFM) system with a multiplexing capacity of at least 1480 microspot binding sites, colorimetric readout, high sensitivity, and assay time of <10 min before imaging and data analysis. \ud \ud METHOD:\ud Affinity binders were deposited on nitrocellulose membranes by conventional microarray printing. Buffers and reagents were applied vertically by use of a flow controlled syringe pump. As a clinical model system, we analyzed 31 precharacterized human serum samples using the array system with 10 allergen components to detect specific IgE reactivities. We detected bound analytes using gold nanoparticle conjugates with assay time of ≤10 min. Microarray images were captured by a consumer-grade flatbed scanner. \ud \ud RESULTS:\ud A sensitivity of 1 ng/mL was demonstrated with the VFM assay with colorimetric readout. The reproducibility (CV) of the system was <14%. The observed concordance with a clinical assay, ImmunoCAP, was R2 = 0.89 (n = 31). \ud \ud CONCLUSIONS:\ud In this proof-of-concept study, we demonstrated that the VFM assay, which combines features from protein microarrays and paper-based colorimetric systems, could offer an interesting alternative for future highly multiplexed affinity point-of-care testing

A lateral flow paper microarray for rapid allergy point of care diagnostics

There is a growing need for multiplexed specific IgE tests that can accurately evaluate patient sensitization profiles. However, currently available commercial tests are either single/low-plexed or require sophisticated instrumentation at considerable cost per assay. Here, we present a novel convenient lateral flow microarray-based device that employs a novel dual labelled gold nanoparticle-strategy for rapid and sensitive detection of a panel of 15 specific IgE responses in 35 clinical serum samples. Each gold nanoparticle was conjugated to an optimized ratio of HRP and anti-IgE, allowing significant enzymatic amplification to improve the sensitivity of the assay as compared to commercially available detection reagents. The mean inter-assay variability of the developed LFM assay was 12% CV, and analysis of a cohort of clinical samples (n = 35) revealed good general agreement with ImmunoCAP, yet with a varying performance among allergens (AUC = [0.54-0.88], threshold 1 kU). Due to the rapid and simple procedure, inexpensive materials and read-out by means of a consumer flatbed scanner, the presented assay may provide an interesting low-cost alternative to existing multiplexed methods when thresholds > 1 kU are acceptable

Towards paper-based point of care affinity proteomics

Affinity-based methods such as protein microarrays have come to complement conventional mass/charge-based techniques for proteomic characterization of biological samples. Simultaneous measurement of hundreds or even thousands of serum biomarkers such as antibodies, antigens and other proteins may greatly improve the diagnostic accuracy in a variety of conditions such as autoimmune disorders, infections and several cancers. However, today's technologies for affinity proteomics are cumbersome, require expensive equipment and skilled operators. Here, we present two novel paper-based techniques developed in our lab that aim to bridge the gap between highly multiplexed affinity proteomics and point of care testing

3-D Microwell Array System for Culturing Virus Infected Tumor Cells

Cancer cells have been increasingly grown in pharmaceutical research to understand tumorigenesis and develop new therapeutic drugs. Currently, cells are typically grown using two-dimensional (2-D) cell culture approaches, where the native tumor microenvironment is difficult to recapitulate. Thus, one of the main obstacles in oncology is the lack of proper infection models that recount main features present in tumors. In recent years, microtechnology-based platforms have been employed to generate three-dimensional (3-D) models that better mimic the native microenvironment in cell culture. Here, we present an innovative approach to culture Kaposi’s sarcoma-associated herpesvirus (KSHV) infected human B cells in 3-D using a microwell array system. The results demonstrate that the KSHV-infected B cells can be grown up to 15 days in a 3-D culture. Compared with 2-D, cells grown in 3-D had increased numbers of KSHV latency-associated nuclear antigen (LANA) dots, as detected by immunofluorescence microscopy, indicating a higher viral genome copy number. Cells in 3-D also demonstrated a higher rate of lytic reactivation. The 3-D microwell array system has the potential to improve 3-D cell oncology models and allow for better-controlled studies for drug discovery