Universal Nanoparticle Counting Platform for Tetraplexed Biomarkers by Integrating Immunorecognition and Nucleic Acid Hybridization in One Assay.

The development of a simple and universal strategy for simultaneous quantification of proteins and nucleic acid biomarkers in one assay is valuable, particularly for disease diagnosis and pathogenesis studies. Herein, a universal and amplification-free quantum dot-doped nanoparticle counting platform was developed by integrating immunorecognition and nucleic acid hybridization in one assay. The assay can be performed at room temperature, which is friendly for routine analysis. Multiplexed biomarkers associated with Alzheimer's disease (AD) including proteins and nucleic acids were detected. For simultaneous detection of tetraplex biomarkers, the assay for amyloid ß 1-42 (Aß42), tau protein, miR-146a, and miR-138 presented limit of detection values of 250 pg/mL, 55.7 pg/mL, 52.5 pM, and 0.62 pM, respectively. By spiking all the above four biomarkers in one artificial cerebrospinal fluid sample, the recoveries were found to be 94.7-117.2%. Using tau protein as the model, four measurements in 88 days presented a coefficient of variance of 7.5%. The proposed platform for the multiplexed assay of proteins and nucleic acids presents the universality, reasonable sensitivity, and repeatability, which may open a new door for early diagnosis and pathogenesis research for AD and other diseases.

Aggregation-Enhanced Energy Transfer for Mitochondria-Targeted ATP Ratiometric Imaging in Living Cells.

Förster resonance energy transfer (FRET) from fluorescent nanoparticles to fluorescent dyes is an attractive approach for bioanalysis in living cells. However, the luminescence of the nanoparticle donor/acceptor has not been effectively used to produce highly efficient FRET because the distance between the energy donor and energy acceptor is often larger than the effective FRET radius (about 10 nm) and the uncontrolled rotational and translational diffusion of luminophores. Here, we develop an aggregation-enhanced energy transfer strategy that can overcome the impedance for effective energy transfer. The functional nanoprobes, named TPP-CDs-FITC, are carbon dots (CDs) functionalized with triphenylphosphine (TPP) and ∼117 fluorescein 5-isothiocyanate (FITC) on the surface. In dispersed solution, the 3.8 nm TPP-CDs-FITC show weak FRET efficiency (15.4%). After TPP-instructed mitochondrial targeting, enhanced FRET efficiency (53.2%) is induced due to the aggregation of TPP-CDs-FITC selectively triggered by adenosine triphosphate (ATP) in the mitochondria. The enhanced FRET efficiency can be attributed to the joint effect of the augment of numbers of FITC acceptors within 10 nm from dispersed 117 to aggregated 5499 and the restricted rotational and translational motions of TPP-CDs donors and FITC acceptors. Ultimately, we successfully observe the fluctuations of ATP levels in the mitochondria using the aggregation-enhanced energy transfer strategy of the TPP-CDs-FITC nanodevice.

Colocalized Particle Counting Platform for Zeptomole Level Multiplexed Quantification.

For multiplexed detection, it is important yet challenging to simultaneously meet the requirement of sensitivity, throughput, and implementation convenience for practical applications. Using the detection of DNAs and miRNAs for illustration, we present a colocalized particle counting platform that can realize the separation-free multiplexed detection of 6 nucleic acid targets with a zeptomole sensitivity and a dynamic range of up to 5 orders of magnitude. The presence of target induces the formation of a sandwich nanostructure via hybridization; thus, there is an occurrence of colocalization of two microbeads with two different colors. The sequence specific coding is realized by an arbitrary combination of two fluorescence channels with different emitting colors. The platform presents robustness in detecting multiple nucleic acid targets with a minimal cross talk and matrix effect as well as the ability to distinguish the specific miRNA from members of the same family. The results of simultaneous detection of 3 miRNAs in 3 different cell lines present straight consistency with that of the standard qRT-PCR. This platform can be adapted to other multiplexing designs such as the "turn-off" mode, in which the proportion of colocalized microbeads is decreased due to the strand-displacement reaction initiated by the specific target. This separation-free platform offers the possibility to achieve the on-site multiplexed detection with compatibility to different experimental designs and extensibility to other signal sources for enumeration.

An on-site bacterial detection strategy based on broad-spectrum antibacterial ε-polylysine functionalized magnetic nanoparticles combined with a portable fluorometer.

A sensitive on-site bacterial detection strategy is presented that integrates the broad-spectrum capturing feature of ε-polylysine-functionalized magnetic nanoparticles with an in-house built portable fluorometer. Based on the electrostatic interaction, the functionalized magnetic nanoparticles (ε-PL-MNPs) were prepared for Gram-positive and Gram-negative bacterial separation and subsequent viable release. ε-PL-MNPs show a broad reactivity towards bacteria with the high capture efficiency from real-world sample media. They also enable controlled viable bacterial release with pH adjustment. Detection of bacteria is based on a combination of broad-spectrum capture with colorimetric and fluorimetric immunoassays. A portable fluorometer is built to enhance the applicability for sensitive on-site detection. A limit of detection of 98 CFU·mL-1 is achieved that is comparable to that of a known spectrofluorometric method for E. coli DH5α. Graphical abstract Schematic presentation of bacterial capture using cationic polymer functionalized magnetic nanoparticles and general fluorometric immunoassay with portable fluorometer. The limit of detection is 98 CFU·mL-1 for E. coli DH5α.

Ultraspecific Multiplexed Detection of Low-Abundance Single-Nucleotide Variants by Combining a Masking Tactic with Fluorescent Nanoparticle Counting.

To be able to detect simultaneously multiple single-nucleotide variants (SNVs) with both ultrahigh specificity and low-abundance sensitivity is of pivotal importance for molecular diagnostics and biological research. In this contribution, we for the first time developed a multiplex SNV detection method that combines the masking tactic with fluorescent nanoparticle (FNP) counting based on the sandwich design. The method presents a rivaling performance due to its advantageous features: the masking reagent was designed to hybridize with an extremely large amount of the wild-type sequence to render the assay with high specificity; FNP counting provides a sensitive multiplexed SNV detection; the sandwich design facilitates an easy separation to make the detection free of interferences from the matrix. For single SNV target discrimination, including the 6 most frequently occurring DNA KRAS gene mutations and 2 possible RNA KRAS gene mutations as well as 11 artificial mutations, the discrimination factor ranged from 204 to 1177 with the median being 545. Among the tested 19 SNVs, abundances as low as 0.05% were successfully identified in 14 cases, and an abundance as low as 0.1% was identified for the remaining 5 cases. For multiplexed detection of SNVs in the KRAS gene, abundances as low as 0.05-0.1% were achieved for multiple SNVs occurring at the same and different codons. As low as 0.05% low-abundance detection sensitivity was also achieved for PCR amplicons of human genomic DNA extracted from cell samples. This proposed method presents the potential for ultrahigh specific multiplexed detection of SNVs with low-abundance detection capability, which may be applied to practical applications.

Multiplexed Detection of Attomoles of Nucleic Acids Using Fluorescent Nanoparticle Counting Platform.

The sensitive multiplexed detection of nucleic acids in a single sample by a simple manner is of pivotal importance for the diagnosis and therapy of human diseases. Herein, we constructed an automatic fluorescent nanoparticle (FNP) counting platform with a common fluorescence microscopic imaging setup for nonamplification multiplexed detection of attomoles of nucleic acids. Taking the advantages of the highly bright, multicolor emitting FNPs and magnetic separation, the platform enables sensitive multiplexed detection without the need for extra fluorescent labels. Quantification for multiplex DNAs, multiplex microRNAs (miRNA), as well as a DNA and miRNA mixture was achieved with a similar dynamic range, a limit of detection down to 5 amol (5 µL detection volume), and a 81-115% spike recovery from different biological sample matrices. In particular, the sensitivity for multiplex miRNA is by far among the highest without using amplification or the lock nucleic acid hybridization enhancement strategy. Results regarding miRNA-141 from four different cell lines were agreeable with those of the quantitative reverse transcription polymerase chain reaction. Simultaneous detection of miRNA-141 and miRNA-21 in four different cell lines yielded consistent results with publications, indicating the potential for monitoring multiplex miRNA expression associated with the collaborative regulation of important cellular events. This work expands the rule set of multiplex nucleic acid detection strategies and shows promising potential application in clinical diagnosis.