Mix-and-read method for assessment of milk pasteurization using a smartphone or a common digital camera.

Alkaline phosphatase (ALP) is the most widely used marker of the adequacy of milk pasteurization since it is inactivated at temperatures slightly higher than those required for elimination of pathogens. The cutoff level is 350 mU/L. The approved colorimetric, fluorometric, and chemiluminometric methods require specialized readers with photomultipliers as detectors, and the samples are usually analyzed one-by-one. We developed a low-cost mix-and-read method that exploited a smartphone or a common digital camera as detectors for the chemiluminometric determination of ALP in milk. As samples, we used pasteurized cow and sheep milk spiked with ALP, as well as mixtures of pasteurized and raw (non-pasteurized) milk. Chemiluminescence images acquired by the smartphone or the digital camera were analyzed by the ImageJ software. The limits of detection (LODs), for images captured by the smartphone, were 4.4 mU/L and 11.1 mU/L for cow milk and sheep milk, respectively, while with the digital camera, the respective LODs were 6.2 mU/L and 6.7 mU/L, respectively. The coefficients of variation (CVs) at the cutoff level of 350 mU/L were 8% and 8.5% for the cow and sheep milk, respectively. For images by the digital camera, the CVs were 5.8% and 5% for cow and sheep milk, respectively. The performance of the method is similar to methods that use a microtiter plate and a luminometer for chemiluminescence measurements. Sample pretreatment is not necessary. The microtiter well format combined with detection by a smartphone enables the analysis of multiple samples simultaneously. It is anticipated that the method will prove useful for the rapid assessment of milk pasteurization efficiency in dairy industries, especially in remote areas where expensive instruments are not available. Graphical abstract.

Smartphone-based chemiluminometric hybridization assays and quantitative competitive polymerase chain reaction.

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 × 105 copies of target DNA.

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.