Magneto-nanosensor smartphone platform for the detection of HIV and leukocytosis at point-of-care.

The advent of personalized medicine has brought an increased interest in personal health among general consumers. As a result, there is a great need for user-centric point-of-care (POC) health devices. Such devices are equally pertinent in developing countries or resource-limited settings for performing diagnostic tests. However, current POC tests for diseases such as human immunodeficiency virus (HIV) or leukocytosis do not provide adequate levels of sensitivity or do not exist at all. Here, we extend our mobile magneto-nanosensor platform to point-of-care HIV and leukocytosis detection. The platform can be multiplexed, and the circuitry enables portability and sensitivity in the POC setting. A smartphone application simplifies operation and provides guidance to facilitate self-testing. Commercially available POC test kits typically provide only qualitative or semi-quantitative results of a single analyte. The magneto-nanosensor platform can provide users with pleasant user-experience while demonstrating robust sensitive and specific multiplexed quantification and detection of common diseases.

Multidimensional Normalization to Minimize Plate Effects of Suspension Bead Array Data.

Enhanced by the growing number of biobanks, biomarker studies can now be performed with reasonable statistical power by using large sets of samples. Antibody-based proteomics by means of suspension bead arrays offers one attractive approach to analyze serum, plasma, or CSF samples for such studies in microtiter plates. To expand measurements beyond single batches, with either 96 or 384 samples per plate, suitable normalization methods are required to minimize the variation between plates. Here we propose two normalization approaches utilizing MA coordinates. The multidimensional MA (multi-MA) and MA-loess both consider all samples of a microtiter plate per suspension bead array assay and thus do not require any external reference samples. We demonstrate the performance of the two MA normalization methods with data obtained from the analysis of 384 samples including both serum and plasma. Samples were randomized across 96-well sample plates, processed, and analyzed in assay plates, respectively. Using principal component analysis (PCA), we could show that plate-wise clusters found in the first two components were eliminated by multi-MA normalization as compared with other normalization methods. Furthermore, we studied the correlation profiles between random pairs of antibodies and found that both MA normalization methods substantially reduced the inflated correlation introduced by plate effects. Normalization approaches using multi-MA and MA-loess minimized batch effects arising from the analysis of several assay plates with antibody suspension bead arrays. In a simulated biomarker study, multi-MA restored associations lost due to plate effects. Our normalization approaches, which are available as R package MDimNormn, could also be useful in studies using other types of high-throughput assay data.

Development of a three-plex single molecule immunoassay enabling measurement of the EGFR ligands amphiregulin, betacellulin and transforming growth factor α simultaneously in human serum samples.

BACKGROUND: Prior to large studies in breast cancer patients and healthy individuals we established a sensitive three-plex immunoassay to measure the EGFR ligands amphiregulin (AR), betacellulin (BTC) and transforming growth factor α (TGF-α) simultaneously in human serum samples. METHOD: The three-plex immunoassay was developed using single molecule array (Simoa) technology and requires only 20 µL of serum. RESULTS: AR, BTC and TGF-α were first established as three single-plex assays. Multiplexing the three single-plex assays showed no significant cross reactivity between the reagents. The concentrations of the ligands in serum samples showed correlations r2 ≥ 0.84 between the single-plex and three-plex methods. The three-plex assay demonstrated limit of detection levels at 0.16 ng/L for AR, 0.23 ng/L for BTC and 0.22 ng/L for TGF-α. Total coefficients of variations were 8.5%-31% for AR, 11%-21.8% for BTC and 12.4%-16.2% for TGF-α. Spiking experiments showed a mean recovery of 97% for AR, 86% for BTC and 81% for TGF-α. The concentrations of the EGFR ligands did not change significantly after series of freeze thaw cycles or incubation at 22 °C for up to 24 h. CONCLUSION: This robust three-plex assay with up to 40-fold increase in sensitivity relative to conventional ELISA is the first published method that has the required sensitivity to measure AR, BTC and TGF-α simultaneously in human blood samples.

Giant magnetoresistive sensor array for sensitive and specific multiplexed food allergen detection.

Current common allergen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and dip-stick methods, do not provide adequate levels of sensitivity and specificity for at-risk allergic patients. A method for performing highly sensitive and specific detection of multiple food allergens is thus imperative as food allergies are becoming increasingly recognized as a major healthcare concern, affecting an estimated 4% of the total population. We demonstrate first instance of sensitive and specific multiplexed detection of major peanut allergens Ara h 1 and Ara h 2, and wheat allergen Gliadin using giant magnetoresistive (GMR) sensor arrays. Commercialized ELISA kits for Ara h 1 and Ara h 2 report limits of detection (LODs) at 31.5 ng/mL and 0.2 ng/mL, respectively. In addition, the 96-well-based ELISA developed in-house for Gliadin was found to have a LOD of 40 ng/mL. Our multiplexed GMR-based assay demonstrates the ability to perform all three assays on the same chip specifically and with sensitivities at LODs about an order of magnitude lower than those of 96-well-based ELISAs. LODs of GMR-based assays developed for Ara h 1, Ara h 2, and Gliadin were 7.0 ng/mL, 0.2 ng/mL, and 1.5 ng/mL, respectively, with little to no cross-reactivity. These LODs are clinically important as some patients could react strongly against such low allergen levels. Given the limitations of current industrial detection technology, multiplexed GMR-based assays provide a method for highly sensitive and specific simultaneous detection of any combination of food-product allergens, thus protecting allergic patients from life-threatening events, including anaphylaxis, by unintentional consumption.

A fully-automated, six-plex single molecule immunoassay for measuring cytokines in blood.

We report a system and assay for performing fully-automated measurement of 6 proteins simultaneously with single molecule sensitivity. The system combines handling of samples, reagents, and consumables, with a module for imaging single molecule arrays (Simoa) to enable immunoassays that have high sensitivity (~fg/mL), are multiplexed, and are fully-automated. A 6-plex cytokine Simoa assay for IL-6, TNF-α, GM-CSF, IL-10, IL-1ß, and IL-1α was developed on the system. The assays had limits of detection in the range 0.01-0.03pg/mL, and the average imprecision (CV) of the Simoa signal was 4.2%. This assay was used to measure the concentrations of these cytokines in the plasma of patients with Crohn's Disease (CD), before and after treatment with anti-TNF-α antibody drugs, and in the serum of Type 1 diabetics. Concentrations of TNF-α and IL-6 in the CD samples determined using the fully-automated, multiplex Simoa assay had good correlation with the manual, single-plex assays previously reported. Drug treatment caused reductions in the mean concentration of all 6 cytokines in the plasma of CD patients. The concentrations of 4 cytokines were significantly higher in diabetics compared to healthy controls. The system could enable the widespread, multiplexed measurement of protein biomarkers with low abundance.

CuO-induced signal amplification strategy for multiplexed photoelectrochemical immunosensing using CdS sensitized ZnO nanotubes arrays as photoactive material and AuPd alloy nanoparticles as electron sink.

In this work, multiplexed photoelectrochemical (PEC) immunoassays are introduced into an indium tin oxide (ITO) device. Firstly, the ITO device is fabricated using a simple acid etch treatment method. Secondly, AuPd alloy nanoparticles are electro-deposited on ITO working electrodes as electron sink to construct the immunosensor platform. After that, ZnO nanotubes (ZNTs) arrays are synthesized via chemical etching of ZnO nanorods that are grown on AuPd surface by electrochemical deposition method. Subsequently, CdS is electro-deposited on ZNTs arrays and used as photoactive material. Then, CuO nanoseeds are labeled with signal antibodies and firstly used as PEC signal amplification label. The introduction of CuO brings signal amplification because of the conduction band (CB) of both CuO and ZnO are lower than that of CdS, CuO will compete the photo-induced electrons in CB of CdS with ZnO, leading to the decrease of the photocurrent intensity. Using cancer antigen 125, prostate specific antigen and α-fetoprotein as model analytes, the proposed immunoassay exhibits excellent precision and sensitivity. Meanwhile, this work provides a promising, addressable and simple strategy for the multi-detection of tumor markers.