Immunomagnetic Capture Research Articles

Abstract 293: Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression

Abstract Breast Cancer (BC) is a heterogeneous disease that impacts treatment response. This is due in part to the tumor microenvironment (TME), which contributes significantly to BC pathogenesis, including metastasis. Women with higher breast density and reduced breast fat face an increased BC risk across multiple tumor subtypes. This is exemplified by the correlation between increased tumor progression, the appearance of fibrosis and the loss of mature adipocytes. While the role of adipokines in this process has been studied extensively, the contribution of adipocyte-derived extracellular vesicles (EV) or "adipomes" in tumorigenesis and metastasis remains poorly understood. To address this question, we developed a novel technique for isolating intact adipomes from normal mammary fat (MF) obtained from wild-type C57BL/6 mice as well as from tumor-associated MF (TAMF) obtained from mice inoculated orthotopically with EO771 mammary tumor cells. Adipome isolation involved high-speed centrifugation to separate macro- and micro-vesicles, purification using the ExoQuick EV isolation system, and immunomagnetic capture of adipomes. Characterization of adipomes by transmission electron microscopy revealed two adipome populations, large L-adipomes of ~350 nm, and small S-adipomes of ~40 nm. Notably, the quantity of L-adipomes in tumor-associated mammary fat (TAMF) was 10-fold greater than the amount of L-adipomes in the MF of non-tumor-bearing mice. To identify possible functional roles of L-adipomes in the pathogenesis of BC, lipidomic profiling was undertaken. L-Adipomes from TAMF showed significantly altered lipid species in comparison to MF-derived L-adipomes. Adipomes were further characterized by treating EO771 cells for 72 hr with TAMF- or MF-derived adipomes at a cell to adipome ratio of 1:10, followed by RNAseq analysis. Differentially upregulated genes in TAMF-derived L-adipomes included Esr1, Erbb2, Tgfb1, Bcl2, MMP2, and Notch associated with fibrosis, EMT, angiogenesis and invasion and downregulation of genes linked to tumor suppression, apoptosis, and mitochondrial metabolism. Overall, our study illustrates the importance of tumor-associated adipomes in regulating gene expression in tumor cells, which emphasizes the specificity of adipome content relating to the cell-of-origin and the host's pathologic and metabolic state and in modulating signaling pathways involved in BC progression. Citation Format: Hariprasad Thangavel, Kezia Lizardo, Robert I. Glazer, Jyothi F. Nagajyothi. Distinctive roles of mammary adipomes in breast cancer progression: Insights from functional analysis and differential gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 293.

Immunocapture of cell surface proteins embedded in HIV envelopes uncovers considerable virion genetic diversity associated with different source cell types.

HIV particles in the blood largely originate from activated lymphocytes and can overshadow variants which may be expressed from other cell types. Investigations of virus persistence must be able to distinguish cells refractory to viral clearance that serve as reservoirs. To investigate additional cell types that may be associated with in vivo HIV expression we developed a virus particle immunomagnetic capture method targeting several markers of cellular origin that become embedded within virion envelopes during budding. We evaluated the ability of markers to better distinguish cell lineage source subpopulations by assessing combinations of different antibodies with cell-sorted in vitro culture and clinical specimens. Various deductive algorithms were designed to discriminate source cell lineages and subsets. From the particle capture algorithms, we identified distinct variants expressed within individuals that were associated with disparate cellular markers. Among the variants uncovered were minority-level viruses with drug resistance mutations undetected by sequencing and often were associated with markers indicative of myeloid lineage (CD3-/CD10-/CD16+ or /CD14+, and CD3-/CD16-/CD14-/CD11c+ or /HLA-DR+) cell sources. The diverse HIV genetic sequences expressed from different cell types within individuals, further supported by the appearance of distinct drug-resistant variants, highlights the complexity of HIV reservoirs in vivo which must be considered for HIV cure strategies. This approach could also be helpful in examining in vivo host cell origins and genetic diversity in infections involving other families of budding viruses.

The integrated on-chip isolation and detection of circulating tumour cells.

Circulating tumour cells (CTCs) are cancer cells shed from a primary tumour which intravasate into the blood stream and have the potential to extravasate into distant tissues, seeding metastatic lesions. As such, they can offer important insight into cancer progression with their presence generally associated with a poor prognosis. The detection and enumeration of CTCs is, therefore, critical to guiding clinical decisions during treatment and providing information on disease state. CTC isolation has been investigated using a plethora of methodologies, of which immunomagnetic capture and microfluidic size-based filtration are the most impactful to date. However, the isolation and detection of CTCs from whole blood comes with many technical barriers, such as those presented by the phenotypic heterogeneity of cell surface markers, with morphological similarity to healthy blood cells, and their low relative abundance (∼1 CTC/1 billion blood cells). At present, the majority of reported methods dissociate CTC isolation from detection, a workflow which undoubtedly contributes to loss from an already sparse population. This review focuses on developments wherein isolation and detection have been integrated into a single-step, microfluidic configuration, reducing CTC loss, increasing throughput, and enabling an on-chip CTC analysis with minimal operator intervention. Particular attention is given to immune-affinity, microfluidic CTC isolation, coupled to optical, physical, and electrochemical CTC detection (quantitative or otherwise).

Integration of protein L-immobilized epoxy magnetic bead capture with LC-MS/MS for therapeutic monoclonal antibody quantification in serum.

In recent years, there has been a growing interest in the thriving monoclonal antibody (mAb) industry due to the wide utilization of mAbs in clinical therapies. Robust and accurate bioanalytical methods are required to enable fast quantification of mAbs in biological matrices, especially in the context of pharmacokinetics (PKs)/pharmacodynamics (PDs) and therapeutic drug monitoring (TDM) studies. In this investigation, we presented a novel immuno-magnetic capture coupled with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed for the quantification of immunoglobulin G-kappa-based mAbs in biological fluids. The immunoaffinity absorbent for mAb drug purification was meticulously crafted by immobilizing protein L onto monosize, magnetic poly(glycidyl methacrylate) (m-pGMA) beads, synthesized through dispersion polymerization. The microspheres were acquired with an average size of 1.6 μm, and the optimal binding of mAbs from the aqueous mAb solution was determined to be 45.82 mg g-1. The quantification of mAbs in 10 μL serum samples was achieved through affinity purification using m-pGMA@protein L beads (employing rituximab as an internal standard (IS)), on-bead reduction, and rapid tryptic digestion. Remarkably, the entire process, taking less than 2.5 hours, held significant potential for simplifying pretreatment procedures and minimizing analytical time. Furthermore, the developed method underwent validation in accordance with the European Medicines Agency (EMA) guidelines. The assay demonstrated commendable linearity within the 2-400 μg mL-1 range for both daratumumab and pembrolizumab. Intra- and inter-assay coefficients of variation fell within the range of 0.7% to 13.4%, meeting established acceptance criteria. Other validation parameters also conformed to regulatory standards. Ultimately, the efficacy of the method was substantiated in a pharmacokinetic study following a single-dose intravenous administration to mice, underscoring its applicability and reliability in real-world scenarios.

Immunomagnetic capture and traceless release of native tumor-derived exosomes from human plasma for exploring interaction with recipient cells by aptamer-functionalized nanoflowers

BackgroundTumor-derived exosomes (TEXs) play an important role in the development process of cancer, which can transport a large number of carcinogenic molecules to normal cells, and subsequently promote tumor metastasis. However, TEXs that were utilized in most of previous researches were obtained from the cell medium of tumor cell lines, which cannot reflect the physiological state of primary cells in vivo. Isolation of native TEXs from human plasma with intact function is contributed to exploring the interaction between TEXs and recipient cells for understanding their true biological functions. ResultsWe developed a strategy that involves both capture and release processes to obtain native TEXs from plasma of cancer patients. An MoS2-based immunomagnetic probe (Fe3O4@MoS2-Au-Aptamer, named as FMAA) with the advantages of high surface area, magnetic response and abundant affinity sites was designed and synthesized to capture TEXs through recognizing high-expression tumor-associated antigens of EpCAM. With the assistance of complementary sequences of EpCAM, TEXs were released with non-destruction and no residual labels. According to NTA analysis, 107-108 TEXs were recovered from per mL plasma of breast cancer patients. The interaction between native TEXs and normal epithelial cells confirms TEXs could induce significant activation of autophagy of recipient cells with co-culture for 12 h. Proteomics analysis demonstrated a total of 637 proteins inside epithelial cells had dynamic expression with the stimulation of TEXs and 5 proteins in the pathway of autophagy had elevated expression level. SignificanceThis work not only obtains native TEXs from human plasma with non-destruction and no residual labels, but also explores the interaction between TEXs and recipient cells for understanding their true biological functions, which will accelerate the application of TEXs in the field of biomarkers and therapeutic drugs.

Electrochemical Detection of SARS-CoV-2 Using Immunomagnetic Separation and Gold Nanoparticles on Unmodified Screen-Printed Carbon Electrodes

The COVID-19 pandemic has underscored the critical need for virus detection methods that are precise, simple, quick, and cost-effective. Electrochemical immunoassay-based methods are a practical solution given their ability to quickly, inexpensively, sensitively, and selectively detect the virus at the point of care. This study details the immunomagnetic capture of SARS-CoV-2 nucleocapsid protein in nasal samples, followed by electrochemical detection using gold nanoparticle labels on a screen-printed carbon electrode. We determined ideal conditions for the size of the gold nanoparticles and the length of the deposition time to maximize the electrochemical signal. The limit of detection for nucleocapsid protein was determined to be 2.64 ng/mL in PBS. The assay was successfully demonstrated to detect nucleocapsid protein in SARS-CoV-2-positive samples with a viral load as low as Ct = 25 (p-value < 0.0001 vs. negative patient control).

Presence of Circulating Tumor Cells Predates Imaging Detection of Relapse in Patients with Stage III Melanoma.

Stage III melanoma includes nodal metastasis or in-transit disease. Five-year survival rates vary between 32% and 93%. The identification of high-risk patients is important for clinical decision making. We demonstrated previously that ≥1 circulating tumor cells (CTCs) at baseline was associated with recurrence. In this study, we investigated how frequently CTCs were identified prior to radiologically detected recurrence. Stage III patients (n = 325) had imaging at baseline and q 3 months. Baseline and q 6-12 months blood draws (7.5 mL) were performed to identify CTCs up to 3.5 years from diagnosis. CTC assessment was performed using the immunomagnetic capture of CD146-positive cells and anti-MEL-PE. The presence of one or more CTCs was considered positive. We analyzed the cohort of patients with relapse confirmed by radiologic imaging. CTC collection dates were assessed to determine the lead time for CTC detection. CTC-negative patients were significantly less likely to relapse compared to patients positive for CTCs (p-value < 0.001). Within the 325-patient cohort, 143 patients (44%) had recurrence, with a median follow-up of 52 months from diagnosis. The cohort (n = 143) with positive imaging and CTC results revealed 76% of patients (108/143) had CTC+ results before the radiological identification of relapse. The median time between positive CTC and positive imaging was 9 months. CTCs were positive in >75% of patients prior to relapse at a median of 9 months before radiologic detection.

Immunomagnetic-bead enriched culturomics (IMBEC) for isolating pathobionts from feces of colorectal cancer patients.

Culturomics employs various cultivating conditions to obtain different types of bacteria and new species. However, current culturomics lacks a highly efficient method for isolating specific pathobionts. Immunomagnetic bead technology, which uses magnetic beads conjugated with antibodies for capturing the antigen to realize enrichment of the targets, has been employed as an alternative method. In this study, we developed a novel method, immunomagnetic bead-enriched culturomics (IMBEC), in which magnetic bead-conjugated antibodies purified from the fecal samples of patients with colorectal cancer (CRC) were used to enrich and isolate potential pathobionts. A protocol for enriching potential pathobionts via immunomagnetic capture was developed by optimizing the concentrations of coupling reagents, NaCl, and detergent. The efficacy of pathobiont enrichment was compared between antibody-coated magnetic beads (antibody group) and nonconjugated blank magnetic beads (blank group). To determine the proinflammatory potential of isolates from both groups, we investigated their ability to induce cytokine production in THP-1 macrophages. This protocol was employed for isolating bacteria from 10 fecal samples of patients with CRC, which were simultaneously compared with those isolated from the blank group. A total of 209 bacterial species were isolated from both groups, including 173 from the antibody group, 160 from the blank group, and 124 from both groups. Bacteria isolated from the antibody group produced more proinflammatory cytokines than those isolated from the blank group. IMBEC is a promising method for relatively specific isolation of potential pathobionts for a particular disease of interest.

Targeting Mucin Protein Enables Rapid and Efficient Ovarian Cancer Cell Capture: Role of Nanoparticle Properties in Efficient Capture and Culture.

The development of specific and sensitive immunomagnetic cell separation nanotechnologies is central to enhancing the diagnostic relevance of circulating tumor cells (CTCs) and improving cancer patient outcomes. The limited number of specific biomarkers used to enrich a phenotypically diverse set of CTCs from liquid biopsies has limited CTC yields and purity. The ultra-high molecular weight mucin, mucin16(MUC16) is shown to physically shield key membrane proteins responsible for activating immune responses against ovarian cancer cells and may interfere with the binding of magnetic nanoparticles to popular immunomagnetic cell capture antigens. MUC16 is expressed in ≈90% of ovarian cancers and is almost universal in High Grade Serous Epithelial Ovarian Cancer. This work demonstrates that cell bound MUC16 is an effective target for rapid immunomagnetic extraction of expressor cells with near quantitative yield, high purity and viability from serum. The results provide a mechanistic insight into the effects of nanoparticle physical properties and immunomagnetic labeling on the efficiency of immunomagnetic cell isolation. The growth of these cells has also been studied after separation, demonstrating that nanoparticle size impacts cell-particle behavior and growth rate. These results present the successful isolation of "masked" CTCs enabling new strategies for the detection of cancer recurrence and select and monitor chemotherapy.

Circulating Endothelial Cells: A New Possible Marker of Endothelial Damage in Kawasaki Disease, Multisystem Inflammatory Syndrome in Children and Acute SARS-CoV-2 Infection.

Background: Kawasaki Disease (KD) and Multisystem Inflammatory Syndrome in Children (MIS-C) are pediatric diseases characterized by systemic inflammation and vascular injury, potentially leading to coronary artery lesions (CALs). Data on vascular injury occurring during acute COVID-19 (AC19) in children are still lacking. The aim of our study was to investigate endothelial injury in KD-, MIS-C- and AC19-dosing circulating endothelial cells (CECs). Methods: We conducted a multicenter prospective study. CECs were enumerated by CellSearch technology through the immunomagnetic capture of CD146-positive cells from whole blood. Results: We enrolled 9 KD, 20 MIS-C and 10 AC19. During the acute stage, the AC19 and KD patients had higher CECs levels than the MIS-C patients. From the acute to subacute phase, a significant CEC increase was observed in the KD patients, while a mild decrease was detected in the MIS-C patients. Cellular clusters/syncytia were more common in the KD patients. No correlation between CECs and CALs were found in the MIS-C patients. The incidence of CALs in the KD group was too low to investigate this correlation. Conclusions: Our study suggests a possible role of CECs as biomarkers of systemic inflammation and endothelial dysfunction in KD and MIS-C and different mechanisms of vascular injury in these diseases. Further larger studies are needed.

Research Development on Exosome Separation Technology.

Exosomes are special extracellular vesicles secreted by cells, which are of great significance in the basic research of life science and clinical application and has become a hot research field with rapid development in recent 10years. Therefore, the isolation and separation of exosomes is particularly important for the research and application of exosomes. This paper aims to review the research progress of exosome isolation and separation methods in recent years, including ultracentrifugation, ultrafiltration, size‑exclusion chromatography, precipitation, immunomagnetic bead capture method, aptamer-based isolation, and isolation methods based on microfluidic technology. It is generally accepted that most of the existing methods have limitations, for example, ultracentrifugation is time-consuming and laborious, and immunomagnetic bead capture method and aptamer-based separation method have small sample processing capacity and high cost. As a result, we also introduce some common situations in which two or more methods are combined for use. Finally, the separation and isolation methods including all those presented in this review were compared and summarized.

Cross-reactivity of antibodies to different rumen methanogens demonstrated using immunomagnetic capture technology.

Methane is produced in the rumen of ruminant livestock by methanogens, accounting for approximately 14.5% of anthropogenic greenhouse gas emissions in terms of global warming potential. The rumen contains a diversity of methanogens species, and only a few of these have been cultured. Immunomagnetic capture technology (ICT) is a simple and effective method to capture and concentrate target organisms in samples containing complex microflora. We hypothesized that antibody-coated magnetic beads could be used to demonstrate antibody specificity and cross-reactivity to methanogens in rumen samples. Sheep polyclonal antibodies raised against four isolates of rumen dwelling methanogens, Methanobrevibacter ruminantium strain M1, Methanobrevibacter sp. AbM4, Methanobrevibacter sp. D5, and Methanobrevibacter sp. SM9 or an equal mix of all four isolates, were used to coat paramagnetic beads. ICT was used together with flow cytometry and qPCR to optimize key parameters: the ratio of antibody to beads, coupling time between antibody and paramagnetic beads to produce immunomagnetic beads (IMBs), and optimal incubation time for the capture of methanogen cells by IMBs. Under optimized conditions, IMBs bound strongly to their respective isolates and showed a degree of cross-reactivity with isolates of other Methanobrevibacter spp. in buffer and in rumen fluid, and with resident methanogens in rumen content samples. The evidence provided here indicates that this method can be used to study the interaction of antibodies with antigens of rumen methanogens, to understand antigen cross-reactivity and antibody binding efficiency for the evaluation of antigens used for the development of a broad-spectrum anti-methanogen vaccine for the abatement of methane production.

On-chip circulating tumor cells isolation based on membrane filtration and immuno-magnetic bead clump capture

Isolating rare circulating tumor cells (CTCs) from blood is critical for the downstream analysis that is important in cancer-related research, diagnosis, and medicine, and efforts are ongoing to increase the efficiency and purity of CTC isolation in microfluidics. Reported in this paper is a two-stage integrated microfluidic chip for coarse-to-fine CTC isolation from whole blood. First, blood cells are removed by filtration using a micropore-array membrane, then CTCs and other cells that are trapped in the micropores are peeled off the membrane by a novel release method based on air–liquid interfacial tension, which significantly increases the recovery rate of CTCs. The second stage is CTC capture based on an on-chip dense immuno-magnetic-bead clump, which offers high capture efficiency and purity. Both the micropore filtration and immuno-magnetic-bead capture are validated and optimized experimentally. Overall, the integrated microfluidic chip can realize a recovery rate of 85.5% and a purity of 37.8% for rare cancer cells spiked in whole blood.

Coupling immuno-magnetic capture with LC-MS/MS(MRM) as a sensitive, reliable, and specific assay for SARS-CoV-2 identification from clinical samples.

Recently, numerous diagnostic approaches from different disciplines have been developed for SARS-CoV-2 diagnosis to monitor and control the COVID-19 pandemic. These include MS-based assays, which provide analytical information on viral proteins. However, their sensitivity is limited, estimated to be 5 × 104 PFU/ml in clinical samples. Here, we present a reliable, specific, and rapid method for the identification of SARS-CoV-2 from nasopharyngeal (NP) specimens, which combines virus capture followed by LC–MS/MS(MRM) analysis of unique peptide markers. The capture of SARS-CoV-2 from the challenging matrix, prior to its tryptic digestion, was accomplished by magnetic beads coated with polyclonal IgG-α-SARS-CoV-2 antibodies, enabling sample concentration while significantly reducing background noise interrupting with LC–MS analysis. A sensitive and specific LC–MS/MS(MRM) analysis method was developed for the identification of selected tryptic peptide markers. The combined assay, which resulted in S/N ratio enhancement, achieved an improved sensitivity of more than 10-fold compared with previously described MS methods. The assay was validated in 29 naive NP specimens, 19 samples were spiked with SARS-CoV-2 and 10 were used as negative controls. Finally, the assay was successfully applied to clinical NP samples (n = 26) pre-determined as either positive or negative by RT-qPCR. This work describes for the first time a combined approach for immuno-magnetic viral isolation coupled with MS analysis. This method is highly reliable, specific, and sensitive; thus, it may potentially serve as a complementary assay to RT-qPCR, the gold standard test. This methodology can be applied to other viruses as well.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03831-5.

SsDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces

ssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. Binders to the EPEC-TirM were selected upon immunomagnetic capture of bacteria bearing active variants and nanobodies identified with a new ability to strongly bind the new antigen. The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest.

Point-of-care diagnosis of invasive non-typhoidal Salmonella enterica in bloodstream infections using immunomagnetic capture and loop-mediated isothermal amplification

Invasive non-typhoidal salmonellosis is gaining worldwide attention as an emerging disease cluster among bloodstream infections. The disease has the highest burden among immunocompromised and malnourished children in resource-limited areas due to poor access to reliable and rapid diagnostics. Point-of-care (POC) diagnostics are promising for use in such low infrastructure laboratory settings. However, there still remains a major challenge for POC testing to deal with the complexity of blood matrices in rapid detection of an extremely low concentration of blood-borne pathogens. In this work, the challenges were addressed by combining magnetic bead based pathogen concentration and Loop Mediated Isothermal Amplification (LAMP) technology. Sensitivity and performance of the combined approach were determined and compared with a direct PCR method. A direct visual detection strategy, adapted using SYTO-24 DNA intercalating dye, resulted in a limit of detection (LoD) as low as 14 CFU/mL in blood samples with a total analysis time of less than 2 h, including sample preparation. This approach has the potential for wide application as a high-throughput POC testing method to analyze pathogens in clinical, food, feed and environmental samples.

Exosomal Surface Protein Detection with Quantum Dots and Immunomagnetic Capture for Cancer Detection.

Exosomes carry molecular contents reflective of parental cells and thereby hold great potential as a source of biomarkers for non-invasive cancer detection and monitoring. However, simple and rapid exosomal molecular detection remains challenging. Here, we report a facile method for exosome surface protein detection using quantum dot coupled with immunomagnetic capture and enrichment. In this method, exosomes were captured by magnetic beads based on CD81 protein expression. Surface protein markers of interest were recognized by primary antibody and then detected by secondary antibody-conjugated quantum dot with fluorescent spectroscopy. Validated by ELISA, our method can specifically detect different surface markers on exosomes from different cancer cell lines and differentiate cancer exosomes from normal exosomes. The clinical potential was demonstrated with pilot plasma samples using HER2-positive breast cancer as the disease model. The results show that exosomes from HER2-positive breast cancer patients exhibited a five times higher level of HER2 expression than healthy controls. Exosomal HER2 showed strong diagnostic power for HER2-positive patients, with the area under the curve of 0.969. This quantum dot-based exosome method is rapid (less than 5 h) and only requires microliters of diluted plasma without pre-purification, practical for routine use for basic vesicle research, and clinical applications.

A Fluorescence Assay for Exosome Detection Based on Bivalent Cholesterol Anchor Triggered Target Conversion and Enzyme-Free Signal Amplification.

Exosomes are emerging as one of the most promising biomarkers for early disease diagnosis and prognosis. The significant challenges facing the available methods include improving the detection specificity and sensitivity in complex biological samples. Herein, a fluorescence assay was established based on a combination of immunomagnetic separation and a two-step signal amplification strategy for direct isolation and subsequent detection of exosomes. First, immunomagnetic beads capture and enrich the exosomes via antibody-antigen reactions. Second, bivalent cholesterol (BC) anchors spontaneously insert into the lipid bilayer of bead-captured exosomes, forming a "one to many" amplification effect. The simultaneous recognition of the surface protein and the lipid bilayer structure of the exosome significantly eliminates the interference risk from free proteins. The detection of exosomes converts to the detection of BC-anchors. Finally, the sticky end of the BC-anchor acts as the initiator to trigger the enzyme-free DNA circuits for secondary signal amplification. Under the optimal conditions, highly sensitive and selective detection of exosomes was achieved ranging from 5.5 × 103 to 1.1 × 107 particles/μL with a limit of detection of 1.29 × 103 particles/μL. Moreover, this method allows the isolation and quantitative analysis of exosomes in several biological fluids with satisfactory recovery rates (92.25-106.8%). Thus, this approach provides a sensitive, anti-interference platform for isolating and detecting exosomes.

Biotin exposure–based immunomagnetic separation coupled with sodium dodecyl sulfate, propidium monoazide, and multiplex real-time PCR for rapid detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk

In this study, we established a rapid and sensitive method for the detection of viable Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes in milk using biotin-exposure-based immunomagnetic separation (IMS) combined with sodium dodecyl sulfate (SDS), propidium monoazide (PMA), and multiplex real-time PCR (mRT-PCR). We used IMS to lessen the assay time for isolation of target bacteria. We then optimized the coupling conditions and immunomagnetic capture process. The immunoreaction and incubation times for 5 μg of mAb coupled with 500 μg of streptavidin-functionalized magnetic beads using a streptavidin-biotin system were 90 and 30 min, respectively. Treatment with SDS-PMA before mRT-PCR amplification eliminated false-positive outcomes from dead bacteria and identified viable target bacteria with good sensitivity and specificity. The limit of detection of IMS combined with the SDS-PMA-mRT-PCR assay for the detection of viable Salmonella Typhimurium, Staph. aureus, and L. monocytogenes in spiked milk matrix samples was 10 cfu/mL and remained significant even in the appearance of 106 cfu/mL of nontarget bacteria. The entire detection process was able to identify viable bacteria within 9 h. The combination of biotin-exposure-mediated IMS and SDS-PMA-mRT-PCR has potential value for the rapid and sensitive detection of foodborne pathogens.

Detection of Melanoma Cells in Whole Blood Samples Using Spectral Imaging and Optical Clearing

Most cancer deaths are associated with metastases resulting from the spread of circulating tumor cells (CTCs) from the primary tumor to vital organs. The existing methods for detection of CTCs as markers of metastasis progression are time consuming with several steps of sample processing, including red blood cell removal, labeling, immunomagnetic capture and isolation, which can lead to loss of CTCs. Here we introduce a method for detection and identification of CTCs using spectral absorption imaging of melanoma cells and optical clearing of whole blood samples. Verification of this approach was performed using phantoms of human melanoma cells and suspensions of mouse melanoma cells of line B16F10 alone and in mixture with blood. A method for improving detection sensitivity has been demonstrated applying optical clearing of mouse blood using biocompatible chemical agents. The findings suggest that the proposed diagnostic platform has the potential to detect quickly CTCs in whole blood samples from patients with melanoma.