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The present report introduces the smartphone as a simple, low-cost detector/imager for chemiluminometric hybridization assays and quantitative competitive polymerase chain reaction (QCPCR). In QCPCR the amplification products from the target and the competitor DNA have identical sizes but differ in a short sequence flanked by the primers. The products are hybridized with their cognate oligonucleotide probes, captured on microtiter wells and detected via an enzyme-catalyzed chemiluminogenic reaction using the smartphone as a detector/imager. We provide, for the first time, data on: (a) the detectability, analytical range and reproducibility of smartphone-based chemiluminometric hybridization assays of double stranded amplification products, (b) the comparison of smartphone-based detection with a conventional digital camera and a luminometer, and (c) the detectability, analytical range and reproducibility of smartphone-based QCPCR in terms of the number of copies of input target sequences in the sample prior to amplification. The limits of detection of the DNA hybridization assay based on the smartphone, digital camera and luminometer were 1.6, 2.4 and 1 pmol L -1 . Smartphone-based QCPCR showed an analytical range from 137 to 9 × 10 5 copies of target DNA., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene. Using PCR as a synthetic tool, a competitor was prepared for each target sequence containing the same primer binding sites as the target but differing in a short segment to enable discrimination by hybridization. The assay involves multiplex amplification of targets and competitors followed by a multiplex hybridization assay for the 16 amplification products. The assay was performed on optically encoded microspheres with oligonucleotide probes attached to their surface. The microspheres were analyzed rapidly (1 min) by flow cytometry. The signal ratio of the target and cognate competitor is a function of the target copy number in the sample prior to amplification. The multiplexing potential of the proposed method is much higher than real-time PCR and other end-point methods since there are 100 sets of commercially available microspheres.

MicroRNAs (miRNAs) are single-stranded noncoding RNA molecules that act as key regulators of mRNA expression and are emerging biomarkers for disease. Their small size (18-25 nt) presents challenges to molecular recognition, labeling, and signal generation. Recent research activity in this field has aimed at the development of methods for miRNA quantification that combine high detectability, broad dynamic range, practicality, multiplexity, and low cost for prospective applications in diagnostic laboratories. This review article covers the most recent advances in microRNA analysis.

Solid-phase extraction (SPE) and capillary electrophoresis (CE) are on-line coupled via a Tee-split interface, which provides hydrodynamic injection of the SPE eluate by flow splitting. The interface allows sample preconcentration independently from the CE separation and prevents sample matrix and washing solvents from entering the separation capillary. The effect of the Tee-split interface on the CE efficiency was examined using enkephalin peptides as model compounds. Most favorable plate numbers were obtained using a split ratio of 1:40. Breakthrough volume, desorption efficiency and elution volume for the C18 micro SPE column (5 mm x 0.5 mm i.d.) were found to be 750 microL, 65% and 1 microL, respectively. The performance of the complete system was demonstrated by the preconcentration and separation of an enkephalin mixture. Plate numbers up to 120,000 were obtained using a sample volume of 250 microL and a split ratio of 1:40. Enkephalin peak areas were linear (R2 = 0.996) over the 10-1000 ng/mL range. UV absorbance concentration limits of detection (SIN = 3) were about 5 ng/mL. For 250 microL injections of 100 ng/mL, the relative standard deviation (n = 5) of peak area was lower than 10%.