Prototype Smartphone-Based Device for Flow Cytometry with Immunolabeling via Supra-nanoparticle Assemblies of Quantum Dots.

Methods for the detection, enumeration, and typing of cells are important in many areas of research and healthcare. In this context, flow cytometers are a widely used research and clinical tool but are also an example of a large and expensive instrument that is limited to specialized laboratories. Smartphones have been shown to have excellent potential to serve as portable and lower-cost platforms for analyses that would normally be done in a laboratory. Here, we developed a prototype smartphone-based flow cytometer (FC). This compact 3D-printed device incorporated a laser diode and a microfluidic flow cell and used the built-in camera of a smartphone to track immunofluorescently labeled cells in suspension and measure their color. This capability was enabled by high-brightness supra-nanoparticle assemblies of colloidal semiconductor quantum dots (SiO2@QDs) as well as a support vector machine (SVM) classification algorithm. The smartphone-based FC device detected and enumerated target cells against a background of other cells, simultaneously and selectively counted two different cell types in a mixture, and used multiple colors of SiO2@QD-antibody conjugates to screen for and identify a particular cell type. The potential limits of multicolor detection are discussed alongside ideas for further development. Our results suggest that innovations in materials and engineering should enable eventual smartphone-based FC assays for clinical applications.

Red-Emissive Cell-Penetrating Polymer Dots Exhibiting Thermally Activated Delayed Fluorescence for Cellular Imaging.

Fluorescence imaging in living cells is key to understanding many biological processes, yet autofluorescence from the sample can lower sensitivity and hinder high-resolution imaging. Time-gated measurements using phosphorescent metal complexes can improve imaging, at the cost of potential toxicity from the use of heavy metals. Here, we describe orange/red-emitting polymer dots (Pdots) exhibiting thermally activated delayed fluorescence (TADF) for time-gated imaging. Inspired by the cell invasion mechanism of the HIV TAT protein, the Pdots were formed from block copolymers composed of a hydrophilic guanidine-rich block as a cell-penetrating peptide mimic, and a rigid organic semiconductor block to provide efficient delayed fluorescence. These all-organic polymer nanoparticles were shown to efficiently enter HeLa, CHO, and HepG2 cells within 30 min, with cell viabilities remaining high for Pdot concentrations up to 25 mg mL-1. Pdot quantum yields were as high as 0.17 in aerated water, with the Pdot structure effectively shielding the TADF emitters from quenching by oxygen. Colocalization experiments revealed that the Pdots primarily accumulate outside of lysosomes, minimizing lysosomal degradation. When used for fixed cellular imaging, Pdot-incubated cells showed high signal-to-background ratios compared to control samples with no Pdot exposure. Using time-resolved spectroscopy, the delayed emission of the TADF materials was effectively separated from that of both a biological serum and a secondary fluorescent dye.

Photoluminescent Nanoparticles for Chemical and Biological Analysis and Imaging.

Research related to the development and application of luminescent nanoparticles (LNPs) for chemical and biological analysis and imaging is flourishing. Novel materials and new applications continue to be reported after two decades of research. This review provides a comprehensive and heuristic overview of this field. It is targeted to both newcomers and experts who are interested in a critical assessment of LNP materials, their properties, strengths and weaknesses, and prospective applications. Numerous LNP materials are cataloged by fundamental descriptions of their chemical identities and physical morphology, quantitative photoluminescence (PL) properties, PL mechanisms, and surface chemistry. These materials include various semiconductor quantum dots, carbon nanotubes, graphene derivatives, carbon dots, nanodiamonds, luminescent metal nanoclusters, lanthanide-doped upconversion nanoparticles and downshifting nanoparticles, triplet-triplet annihilation nanoparticles, persistent-luminescence nanoparticles, conjugated polymer nanoparticles and semiconducting polymer dots, multi-nanoparticle assemblies, and doped and labeled nanoparticles, including but not limited to those based on polymers and silica. As an exercise in the critical assessment of LNP properties, these materials are ranked by several application-related functional criteria. Additional sections highlight recent examples of advances in chemical and biological analysis, point-of-care diagnostics, and cellular, tissue, and in vivo imaging and theranostics. These examples are drawn from the recent literature and organized by both LNP material and the particular properties that are leveraged to an advantage. Finally, a perspective on what comes next for the field is offered.

Meta-analysis: fecal calprotectin for assessment of inflammatory bowel disease activity.

BACKGROUND: Fecal calprotectin (FC) is a promising biomarker for diagnosis of inflammatory bowel disease (IBD). However, the utility of FC for assessment of IBD activity has yet to be clearly demonstrated. The aim of our study was to evaluate the diagnostic accuracy of FC for differentiating between patients with active IBD and those in remission. METHODS: We systematically searched the databases Medline, Web of Science, Cochrane Library, and EMBASE for eligible studies from December 2013 or earlier that evaluated activity in ulcerative colitis (UC) and Crohn's disease (CD). A hierarchical summary receiver operating characteristic model was performed to calculate the area under the curve to evaluate the overall diagnostic accuracy. The sensitivities and specificities of each commonly applied cutoff value were pooled using a random effects model. RESULTS: We included 13 studies (744 patients with UC and 727 with CD) in the final analysis. The area under the curve values were 0.89 (95% confidence interval, 0.86-0.92), 0.93 (0.89-0.97), and 0.88 (0.83-0.93) in the IBD, UC, and CD groups, respectively. For the IBD group at a cutoff value of 50 µg/g, the pooled sensitivity was 0.92 (0.90-0.94) and specificity 0.60 (0.52-0.67). For a cutoff value at 100 µg/g, the pooled sensitivity was 0.84 (0.80-0.88) and specificity was 0.66 (0.59-0.73). For a cutoff value at 250 µg/g, the pooled sensitivity was 0.80 (0.76-0.84) and specificity was 0.82 (0.77-0.86). CONCLUSIONS: The FC test is a reliable marker for assessing IBD disease activity and may have greater ability to evaluate disease activity in UC than CD.

Validation of methylation-sensitive high-resolution melting (MS-HRM) for the detection of stool DNA methylation in colorectal neoplasms.

BACKGROUND: Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for assaying DNA methylation, but its feasibility for assaying stool in patients with colorectal cancer (CRC) is unknown. METHODS: First, the MS-HRM and methylation-specific PCR (MSP) detection limits were tested. Second, the methylation statuses of SFRP2 and VIM were analyzed in stool samples by MS-HRM, and in matching tumor and normal colon tissues via bisulfite sequencing PCR (BSP). Third, a case-control study evaluated the diagnostic sensitivity and specificity of MS-HRM relative to results obtained with MSP and the fecal immunochemical test (FIT). Finally, the linearity and reproducibility of MS-HRM were assessed. RESULTS: The detection limits of MS-HRM and MSP were 1% and 5%, respectively. The diagnostic sensitivities of MS-HRM (87.3%, 55/63) in stool and BSP in matching tumor tissue (92.1%, 58/63) were highly consistent (κ=0.744). The MS-HRM assay detected 92.5% (37/40) methylation in CRCs, 94.4% (34/36) in advanced adenomas, and 8.8% (5/57) in normal controls. The results of MS-HRM analysis were stable and reliable and showed fairly good linearity for both SFRP2 (P<0.001, R(2)=0.957) and VIM (P<0.001, R(2)=0.954). CONCLUSIONS: MS-HRM shows potential for CRC screening.