Improving the Sensitivity of a Multiplexed Digital Immunoassay Based on Extremely High Bead Analysis Efficiency.

The development of a detection methodology with high sensitivity, stability, and user-friendliness for quantification of proteins at subfemtogram levels is essential for clinical applications such as early screening, disease diagnosis, and monitoring disease progression. A traditional micropartition-based digital enzyme-linked immunosorbent assay (dELISA) results in significant bead loss due to intricate partitioning based on Poisson distribution, multistep reaction operations, nonglobal signal recognition, and reading modes, which have not yet achieved the ultimate detection sensitivity. This study introduces an ultrasensitive multiplexed digital immunoassay with extremely high bead analysis efficiency (HiBeA) through integrating the bead transfer strategy in multistep immunoreaction processing and flow cytometry detection mode.

Revolutionizing the capture efficiency of ultrasensitive digital ELISA an antibody oriented-immobilization strategy.

Bead-based digital ELISA, the most sensitive protein quantification method, has drawn much attention to exploring ultra-low abundance biomarkers in the life sciences and clinical applications. However, its major challenge refers to the low antigen capture efficiency in the immunoreaction process due to the low probability of collision between the deficient concentration of the analytes and the captured antibody-immobilized on the beads. Here, we achieved significantly improved reaction efficiency in the digital signal formation by fixing the orientation of antibodies and revealed the kinetic mechanism for the first time.

Early Diagnosis of Colorectal Cancer Based on Bisulfite-free Site-specific Methylation Identification PCR Strategy: High-Sensitivity, Accuracy, and Primary Medical Accessibility.

Due to its decade-long progression, colorectal cancer (CRC) is most suitable for population screening to achieve a significant reduction in its incidence and mortality. DNA methylation has emerged as a potential marker for the early detection of CRC. However, the current mainstream methylation detection method represented by bisulfite conversion has issues such as tedious operation, DNA damage, and unsatisfactory sensitivity.

Hybrid mRNA Nano Vaccine Potentiates Antigenic Peptide Presentation and Dendritic Cell Maturation for Effective Cancer Vaccine Therapy and Enhances Response to Immune Checkpoint Blockade.

Cancer vaccines combined with immune checkpoint blockades (ICB) represent great potential application, yet the insufficient tumor antigen presentation and immature dendritic cells hinder improved efficacy. Here, a hybrid nano vaccine composed by hyper branched poly(beta-amino ester), modified iron oxide nano adjuvant and messenger RNA (mRNA) encoded with model antigen ovalbumin (OVA) is presented. The nano vaccine outperforms three commercialized reagents loaded with the same mRNA, including Lipofectamine MessengerMax, jetPRIME, and in vivo-jetRNA in promoting dendritic cells' transfection, maturation, and peptide presentation.

MSU crystal deposition contributes to inflammation and immune responses in gout remission.

As a prominent feature of gout, monosodium urate (MSU) crystal deposition induces gout flares, but its impact on immune inflammation in gout remission remains unclear. Using single-cell RNA sequencing (scRNA-seq), we characterize the transcription profiling of peripheral blood mononuclear cells (PBMCs) among intercritical remission gout, advanced remission gout, and normal controls. We find systemic inflammation in gout remission with MSU crystal deposition at the intercritical and advanced stages, evidenced by activated inflammatory pathways, strengthened inflammatory cell-cell interactions, and elevated arachidonic acid metabolic activity.

Emerging advances in nanobiomaterials-assisted chimeric antigen receptor (CAR)-macrophages for tumor immunotherapy.

Adoptive cell immunotherapy, especially chimeric antigen receptor (CAR)-T-cells therapy, has made great progress in the clinical treatment of hematological malignancies. However, restricted by the complex tumor microenvironment, the potential efficiency of T-cell infiltration and activated immune cells are limited, thus failure prevented the progression of the solid tumor. Alternatively, tumor-associated macrophages (TAMs), one sustentacular and heterogeneous cellular population within the tumor microenvironment, are regarded as potential therapeutic targets.

Multiplex Digital Immunoassays with Reduced Pre-partition Reaction Massively Parallel Reagent Delivery on a Bead-Based SlipChip.

The emergence of digital immunoassays has advanced the sensitivity of protein analysis to ultrahigh sensitivity at the attomolar level. However, the background signal generated by the premixing of immunocomplexes and fluorogenic substrates can limit the precise quantification, especially in multiplexed assays. Herein, a bead-based SlipChip (bb-SlipChip) microfluidic device capable of massively parallel two-step sample loading is presented.

A micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads and tyramide signal amplification strategy.

Digital bio-detection has become one of the most appealing methods in recent years due to its excellent performance with ultra-sensitivity in detection of low-abundance targets. Traditional digital bio-detection needs the utilization of micro-chambers for physical isolation of targets, while the recently developed beads-based micro-chamber free one is attracting extensive attention, although there exist the disadvantages of overlaps between positive ("1") and negative ("0") signals as well as the decreased detection sensitivity in multiplexed mode. Here we propose a feasible and robust micro-chamber free digital bio-detection for multiplexed and ultrasensitive immunoassay based on encoded magnetic microbeads (EMMs) and tyramide signal amplification (TSA) strategy.

Joint Application of Multiple Inflammatory Cytokines in Diagnosis of Gout Flare.

Purpose: This study aimed to explore the accuracy for joint application of inflammatory cytokines in diagnosis of gout flare by comparison with peripheral blood cells.

Methods: We collected the clinical data of 96 acute gout patients and 144 remission gout patients, and compared the levels of peripheral blood cells, inflammatory cytokines and blood biochemistry indexes between acute and remission gout. We respectively assessed the area under curves (AUCs) for single and multiple inflammatory cytokines including C-reactive protein (CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and single and multiple peripheral blood cells including platelet (PLT), white blood cell (WBC), percentages of neutrophils (N%), lymphocytes (L%), eosinophils (E%), basophils (B%) in diagnosis of acute gout by receiver operating characteristic (ROC) curve analysis.

Multiplexed and accurate quantification strategy for miRNA based on specific terminal-mediated PCR with equivalent amplification.

MicroRNAs (miRNAs) are recognized as potential biomarkers for the early diagnosis and prognosis of different diseases. Multiplexed and accurate miRNA quantification methods with equivalent detection efficiency are particularly crucial due to their complex biological functions and lack of a unified internal reference gene. Here, a unique multiplexed miRNA detection method, named Specific Terminal-Mediated miRNA PCR (STEM-Mi-PCR), was developed.

Sequence terminus dependent PCR for site-specific mutation and modification detection.

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows.

Cell membrane-camouflaged liposomes and neopeptide-loaded liposomes with TLR agonist R848 provides a prime and boost strategy for efficient personalized cancer vaccine therapy.

Recent advances in bioinformatics and nanotechnology offer great opportunities for personalized cancer vaccine development. However, the timely identification of neoantigens and unsatisfactory efficacy of therapeutic cancer vaccines remain two obstacles for clinical transformation. We propose a "prime and boost" strategy to facilitate neoantigen-based immunotherapy.

Mannose and Hyaluronic Acid Dual-Modified Iron Oxide Enhances Neoantigen-Based Peptide Vaccine Therapy by Polarizing Tumor-Associated Macrophages.

Neoantigen-based cancer vaccine therapy is a breakthrough in the field of immunotherapy. However, it is difficult for vaccines against neoantigens to overcome the immunosuppressive microenvironment, where tumor-associated macrophages (TAMs) play a significant role. Herein, we report an iron oxide nanoparticle modified with hyaluronic acid and mannose to reshape the tumor microenvironment by targeting and repolarizing TAMs from protumor M2 to antitumor M1 phenotype.

Ultrasensitive monitoring of SARS-CoV-2-specific antibody responses based on a digital approach reveals one week of IgG seroconversion.

COVID-19 is still unfolding, while many people have been vaccinated. In comparison to nucleic acid testing (NAT), antibody-based immunoassays are faster and more convenient. However, its application has been hampered by its lower sensitivity and the existing fact that by traditional immunoassays, the measurable seroconversion time of pathogen-specific antibodies, such as IgM or IgG, lags far behind that of nucleic acids.

Bead-Based Multiplexed Droplet Digital Polymerase Chain Reaction in a Single Tube Using Universal Sequences: An Ultrasensitive, Cross-Reaction-Free, and High-Throughput Strategy.

The multiplexed digital polymerase chain reaction (PCR) is widely used in molecular diagnosis owing to its high sensitivity and throughput for multiple target detection compared with the single-plexed digital PCR; however, current multiplexed digital PCR technologies lack efficient coding strategies that do not compromise the sensitivity and signal-to-noise (/) ratio. Hence, we propose a fluorescent-encoded bead-based multiplexed droplet digital PCR method for ultra-high coding capacity, along with the creative design of universal sequences (primer and fluorescent TaqMan probe) for ultra-sensitivity and high / ratios. First, pre-amplification is used to introduce universal primers and universal fluorescent TaqMan probes to reduce primer interference and background noise, as well as to enrich regions of interest in targeted analytes.

Sensitive GlaI digestion and terminal transferase PCR for DNA methylation detection.

Highly sensitive and specific detection of DNA methylation is critical for early diagnosis and therapy of cancer. Herein, we propose a novel bisulfite-free PCR assay based on a GlaI methylation specific digestion and terminal transferase (TdT) extension for the detection of methylated DNA with high sensitivity and specificity, denoted as GlaI-TdT methylation PCR. For GlaI-TdT methylation PCR assay, the methylated CpG site is recognized and cut by GlaI selectively firstly, leading to the generation of product with specific free 3' end.

Breaking through the Poisson Distribution: A compact high-efficiency droplet microfluidic system for single-bead encapsulation and digital immunoassay detection.

Droplet encapsulation of a single cell or bead is widely used in digital detection, single-cell sequencing, and drug screening. However, the encapsulation of particles is totally random restricted by the Poisson distribution. The theoretical possibility of single-particle encapsulation is usually only approximately 10%.

Programmable design of isothermal nucleic acid diagnostic assays through abstraction-based models.

Accelerating the design of nucleic acid amplification methods remains a critical challenge in the development of molecular tools to identify biomarkers to diagnose both infectious and non-communicable diseases. Many of the principles that underpin these mechanisms are often complex and can require iterative optimisation. Here we focus on creating a generalisable isothermal nucleic acid amplification methodology, describing the systematic implementation of abstraction-based models for the algorithmic design and application of assays.

Evolution of "On-Barcode" Luminescence Oxygen Channeling Immunoassay by Exploring the Barcode Structure and the Assay System.

The multiplexed luminescence oxygen channeling immunoassay (multi-LOCI) platform we developed recently that combines conventional LOCI and suspension array technology is capable of realizing facile "mix-and-measure" multiplexed assays without tedious washing steps. However, previous work lacks comprehensive studies of the structure-performance relationship of the host-guest-structured barcode, which may obstruct the evolution and further translation of this exciting new technology to practical applications. Accordingly, this work revealed that polyelectrolyte interlayers played a crucial role in tuning the packing density of guest acceptor beads (ABs).

Emerging Nanoparticle Strategies for Modulating Tumor-Associated Macrophage Polarization.

Immunotherapy has made great progress in recent years, yet the efficacy of solid tumors remains far less than expected. One of the main hurdles is to overcome the immune-suppressive tumor microenvironment (TME). Among all cells in TME, tumor-associated macrophages (TAMs) play pivotal roles because of their abundance, multifaceted interactions to adaptive and host immune systems, as well as their context-dependent plasticity.

Development of a Plasma Biomarker Diagnostic Model Incorporating Ultrasensitive Digital Immunoassay as a Screening Strategy for Alzheimer Disease in a Chinese Population.

Background: The ultrasensitive detection of blood-based biomarkers such as amyloid β (Aβ), tau, and neurofilament light (NFL) has drawn much attention in Alzheimer disease (AD) diagnosis. However, few studies have been conducted in the Chinese population. This study aimed to evaluate the ability of plasma biomarker diagnostic models for AD in the Chinese population based on a novel digital immunoassay technology.

A tailored LNA clamping design principle: Efficient, economized, specific and ultrasensitive for the detection of point mutations.

In the development of personalized medicine, the ultrasensitive detection of point mutations that correlate with diseases is important to improve the efficacy of treatment and guide clinical medication. In this study, locked nucleic acid (LNA) was introduced as an amplification suppressor of a massive number of wild-type alleles in an amplification refractory mutation system (ARMS) to achieve the detection of low-abundance mutations with high specificity and sensitivity of at least 0.1%.