PCB-Based Magnetometer as a Platform for Quantification of Lateral-Flow Assays.

This work presents a proof-of-concept demonstration of a novel inductive transducer, the femtoMag, that can be integrated with a lateral-flow assay (LFA) to provide detection and quantification of molecular biomarkers. The femtoMag transducer is manufactured using a low-cost printed circuit board (PCB) technology and can be controlled by relatively inexpensive electronics. It allows rapid high-precision quantification of the number (or amount) of superparamagnetic nanoparticle reporters along the length of an LFA test strip. It has a detection limit of 10-10 emu, which is equivalent to detecting 4 ng of superparamagnetic iron oxide (Fe3O4) nanoparticles. The femtoMag was used to quantify the hCG pregnancy hormone by quantifying the number of 200 nm magnetic reporters (superparamagnetic Fe3O4 nanoparticles embedded into a polymer matrix) immuno-captured within the test line of the LFA strip. A sensitivity of 100 pg/mL has been demonstrated. Upon further design and control electronics improvements, the sensitivity is projected to be better than 10 pg/mL. Analysis suggests that an average of 109 hCG molecules are needed to specifically bind 107 nanoparticles in the test line. The ratio of the number of hCG molecules in the sample to the number of reporters in the test line increases monotonically from 20 to 500 as the hCG concentration increases from 0.1 ng/mL to 10 ng/mL. The low-cost easy-to-use femtoMag platform offers high-sensitivity/high-precision target analyte quantification and promises to bring state-of-the-art medical diagnostic tests to the point of care.

Evaluation of a nanophosphor lateral-flow assay for self-testing for herpes simplex virus type 2 seropositivity.

Herpes Simplex Virus Type 2 (HSV-2) is a common human pathogen that causes life-long illness. The US prevalence of HSV-2 infection is 11.9% for individuals between 15 and 49 years of age. Individuals with HSV-2 infection are more likely to contract and spread other sexually-transmitted infections. Eighty percent of individuals with HSV-2 are unaware of their infection, in part because of the social stigma associated with in-clinic testing for sexually-transmitted infections. We conducted an initial evaluation of a prototype smartphone-based serological lateral-flow immunoassay (LFA) for HSV-2 infection that uses strontium aluminate persistent luminescent nanoparticles (nanophosphors) as reporters. When applied to a test panel of 21 human plasma/serum samples varying in anti-HSV titer, the nanophosphor HSV-2 LFA had 96.7% sensitivity and 100% specificity for detection of HSV-2 infection. The sensitivity of the nanophosphor HSV-2 LFA was higher than that of commercially-available rapid HSV-2 assays tested with the same panel. Analysis of the iPhone nanophosphor HSV-2 LFA strip images with our custom smartphone app gave greater reproducibility compared to ImageJ analysis of strip images. The smartphone-based nanophosphor LFA technology shows promise for private self-testing for sexually-transmitted infections (STI).

Correction: Enhancement of lateral flow assay performance by electromagnetic relocation of reporter particles.

[This corrects the article DOI: 10.1371/journal.pone.0186782.].

Enhancement of lateral flow assay performance by electromagnetic relocation of reporter particles.

Lateral flow assays (LFAs) are a widely-used point-of care diagnostic format, but suffer from limited analytical sensitivity, especially when read by eye. It has recently been reported that LFA performance can be improved by using magnetic reporter particles and an external magnetic field applied at the test line. The mechanism of sensitivity/performance enhancement was suggested to be concentration/retardation of reporter particles at the test line. Here we demonstrate an additional mechanism of particle relocation where reporter particles from the lower depths of the translucent LFA strip relocate to more-visible locations nearer to the top surface, producing a more visible signal. With a magnetic field we observed an improvement in sensitivity of human chorionic gonadotropin (hCG) detection from 1.25 ng/mL to 0.31 ng/mL. We also observed an increase of the color intensity per particle in test lines when the magnetic field was present.

Orientational binding modes of reporters in a viral-nanoparticle lateral flow assay.

Using microscopy and image analysis, we characterize binding of filamentous viral nanoparticles to a fibrous affinity matrix as models for reporter capture in a lateral flow assay (LFA). M13 bacteriophage (M13) displaying an in vivo-biotinylated peptide (AviTag) genetically fused to the M13 tail protein p3 are functionalized with fluorescent labels. We functionalize glass fiber LFA membranes with antibodies to M13, which primarily capture M13 on the major p8 coat proteins, or with avidin, which captures M13 at the biotin-functionalized tail, and compare orientational modes of reporter capture for the side- versus tip-binding recognition interactions. The number of captured M13 is greater for side-binding than for tip-binding, as expected from the number of recognition groups. Whereas two-thirds of side-bound M13 captured by an anti-M13 antibody bind immediately after colliding with the membrane, tip-bound M13 prominently exhibit three additional orientational modes that require M13 to reorient to enable binding. These results are consistent with the idea that the elongated M13 shape couples with the complex flow field in an open and disordered fibrous LFA membrane to enhance capture.

Use of ImageJ to recover information from individual cells in a G protein-coupled receptor assay.

Live-cell assays used in GPCR research often rely on fluorescence techniques that generate large amounts of raw image data. Consequently, the capacity to accurately and timely extract useful information from image and video data has become more and more important. Image J is an open-source program that provides powerful tools with a simple interface designed to fit the needs of image analysis of most researchers. In this chapter, Image J routines to extract information from individual cells in a calcium GPCR assay are described. In these routines, individual cells in the same image/video data can be separated using either a progressive threshold or a local threshold method. Both methods can be optimized to either a maximum number of selection or maximum area selected resulting in conceptually distinct selections.

Towards the miniaturization of GPCR-based live-cell screening assays.

G protein-coupled receptors (GPCRs) play a key role in many physiological or disease-related processes and for this reason are favorite targets of the pharmaceutical industry. Although ~30% of marketed drugs target GPCRs, their potential remains largely untapped. The discovery of new leads calls for the screening of thousands of compounds with high-throughput cell-based assays. Although microtiter plate-based high-throughput screening platforms are well established, microarray and microfluidic technologies hold potential for miniaturization, automation, and biosensor integration that may well redefine the format of GPCR screening assays. This paper reviews the latest research efforts directed to bringing microarray and microfluidic technologies into the realm of GPCR-based, live-cell screening assays.