Search Results (17)

Background: Colorectal cancer (CRC), a leading cause of cancer-related mortality worldwide, necessitates improved non-invasive diagnostic and monitoring tools. Circulating tumor cells (CTCs), as intact cellular biomarkers in liquid biopsies, offer valuable morphological and genetic information and hold significant clinical potential for early detection, prognosis, therapy monitoring, and drug development. Bio-ferrography is a non-invasive immunomagnetic separation technique that isolates magnetically labeled entities from fluid samples onto a glass substrate via a focused external magnetic field. Methods: This study employs, for the first time, bio-ferrography for isolation, counting, and microscopic characterization of circulating tumor cells (CTCs) expressing the human epidermal growth factor receptor (EGFR) from blood biopsies taken from patients in the hospital. Magnetic beads conjugated with anti-EGFR antibodies were used to selectively capture CTCs from peripheral blood samples of patients with metastatic CRC and other epithelial malignancies. The method enabled both enumeration and microscopic characterization of isolated cells. Results: Preliminary clinical results demonstrate that bio-ferrography achieves a sensitivity of 90% in stage-IV patients and exhibits higher true positive detection rates compared to conventional tumor biomarkers, including carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA). Conclusions: These findings highlight the potential of bio-ferrography as a robust platform for CTC isolation and analysis. Full article

Traditional sample preparation for flow cytometry is often labor-intensive, operator-dependent, and reagent-consuming, limiting its suitability for automated and point-of-care biosensing applications. To address these challenges, this study presents a functional modular microfluidic system integrating immunomagnetic beads (IMBs) to enable automated intracellular immunofluorescence (IF) staining. The modular microfluidic platform is enabled by a dynamically actuated three-dimensional magnetic field that couples with IMBs within a microfluidic reaction chamber, requiring only one-dimensional magnet translation to induce effective three-dimensional bead motion. This magnetic–chip cooperative strategy significantly enhances microscale mixing and cell capture, facilitating automated immunostaining of the radiation biomarker in CD4⁺ cells. Finite element simulations were employed to guide magnetic field design by analyzing magnetic force distributions and identifying key parameters, including magnet material, size, spatial arrangement, and chip–magnet distance. Experimental validation using CD4⁺ cell capture confirmed the effectiveness of the magnetic mixing strategy, revealing ∇B·B as the critical design parameter. An N52 NdFeB magnet (6 mm diameter, 10 mm height) positioned within 2.2 mm of the chamber centerline stably retained IMBs at flow rates below 200 µL/min. Under optimized conditions (magnet translation speed of 8 mm/s and a 15 min mixing duration), a maximum cell capture efficiency of 86% was achieved. Subsequent automated γH2AX IF staining demonstrated a strong linear dose–response relationship (R² > 0.9) in mean fluorescence intensity. This study demonstrates a robust and scalable strategy for automating complex IF staining workflows, highlighting the potential of magnetic-field-assisted microfluidic platforms for biosensing applications requiring reliable intracellular biomarker detection. Full article

Heterogeneous expression of a single surface protein within one cell population can drive major functional differences, yet low-expression subtypes remain difficult to isolate. Conventional tube-based immunomagnetic separation collapses all labelled cells into one positive fraction and thus cannot resolve small differences in marker abundance. Here, we present MagSculptor, a microfluidic platform for high-resolution magnetic fractionation of low-expression EpCAM-defined subtypes within one immunomagnetically labelled population at a time. Arrays of soft-magnetic strips create localized high-gradient zones that map modest differences in bead loading onto distinct capture positions, yielding High (H), Medium (M), Low (L), and Negative (N) fractions. Finite element method simulations of coupled magnetic and hydrodynamic fields quantify the field gradients and define an operating window. Experimentally, epithelial cancer cell lines processed sequentially under identical settings show reproducible subtype partitioning. In a low-EpCAM model (MDA-MB-231), conventional flow cytometry, under standard EpCAM staining conditions, did not yield a robust EpCAM-positive gate, whereas MagSculptor still revealed graded subpopulations. Western blotting confirms a monotonic decrease in EpCAM abundance from H to N, and doxorubicin assays show distinct in vitro drug sensitivities, while viability remains above 95%. MagSculptor thus helps extend immunomagnetic separation from binary enrichment to multi-level isolation of low-expression subtypes and provides a convenient front-end for downstream functional and molecular analyses. Full article

Circulating tumor cells (CTCs) are crucial biomarkers for lung cancer metastasis and recurrence, garnering significant clinical attention. Despite this, efficient and cost-effective detection methods remain scarce. Consequently, there is an urgent demand for the development of highly sensitive CTC detection technologies to enhance lung cancer diagnosis and treatment. This study utilized microspheres and A549 cells to model CTCs, assessing the impact of acoustic field forces on cell viability and proliferation and confirming capture efficiency. Subsequently, CTCs from the peripheral blood of patients with lung cancer were captured and identified using fluorescence in situ hybridization, and the results were compared to the immunomagnetic bead method to evaluate the differences between the techniques. Finally, epidermal growth factor receptor (EGFR) mutation analysis was conducted on CTC-positive samples. The findings showed that acoustic microfluidic technology effectively captures microspheres, A549 cells, and CTCs without compromising cell viability or proliferation. Moreover, EGFR mutation analysis successfully identified mutation types in four samples, establishing a basis for personalized targeted therapy. In conclusion, acoustic microfluidic technology preserves cell viability while efficiently capturing CTCs. When integrated with EGFR mutation analysis, it provides robust support for the precise diagnosis and treatment of lung cancer as well as personalized drug therapy. Full article

Alzheimer’s disease (AD) early screening requires non-invasive, high-sensitivity detection of low-abundance biomarkers in complex biofluids like saliva. In this study, we present a miniaturized, silicon-based electrochemical sensor for sequential detection of two AD salivary biomarkers, lactoferrin (Lf) and amyloid β-protein 1-42 (Aβ_1-42), on a single reusable electrode. The sensor features a three-electrode system fabricated by sputter-coating a quartz substrate with gold (Au) sensing electrodes, which are further modified with gold nanoparticles (AuNPs) to form 3D dendritic structures that enhance surface area and electron transfer. To improve specificity, immunomagnetic beads (MBs) are employed to selectively capture and isolate target biomarkers from saliva samples. These MB–biomarker complexes are introduced into a polydimethylsiloxane chamber aligned with Au sensing electrodes, where a detachable magnet localizes the complexes onto the electrode surface to amplify redox signals. The AuNPs/MBs sensor achieves detection limits of 2 μg/mL for Lf and 0.1 pg/mL for Aβ_1-42, outperforming commercial ELISA kits (37.5 pg/mL for Aβ_1-42) and covering physiological salivary concentrations. After the MBs capture the biomarkers, the sensor can output the result within one minute. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements confirm enhanced electron transfer kinetics on AuNP-decorated surfaces, while linear correlations (R² > 0.95) validate quantitative accuracy across biomarker ranges. The compact and integrated design eliminates reliance on bulky instrumentation and enables user-friendly operation, establishing a promising platform for portable, cost-effective AD screening and monitoring. Full article

A novel approach to protein quantification utilizing a microfluidic platform activated by a magnetic assembly of functionalized magnetic beads around soft magnetic capture centers is presented. Functionalized magnetic beads, known for their high surface area and facile manipulation under external magnetic fields, are injected inside microfluidic channels and immobilized magnetically on the surface of glass-coated soft magnetic microwires placed along the symmetry axis of these channels. A fluorescent (Cy5) immunomagnetic sandwich ELISA is then performed by sequentially flowing the sample and all necessary reagents in the microfluidic channels. Direct protein quantification is performed by magnetically releasing the beads from the microwire and evaluating their fluorescence intensity with the help of a miniature (microfluidic-based) flow cytometer. Measurements of ICAM-1 protein concentration in human blood plasma samples confirm the feasibility of the approach through extensive performance benchmarking. The automation and multiplexing capabilities of the proposed platform further demonstrate its potential for protein quantification in point-of-care settings using microfluidics and miniature flow cytometry instruments. Full article

Introduction: Shiga toxin-producing Escherichia coli (STEC), particularly E. coli O157:H7, is a major contributor to foodborne outbreaks globally. Both E. coli O157 and non-O157 strains can lead to severe health issues, including hemolytic colitis and hemolytic uremic syndrome, which can result in kidney failure. Methods: Two hundred and one frozen beef samples were purchased from various supermarkets located in the Eastern Province of Saudi Arabia and subsequently enriched in tryptic soy broth (TSB). From the enriched samples in TSB, 1 mL portion was mixed with immunomagnetic beads (IMB) coated with specific antibodies targeting the E. coli O157 O antigen. The beads, which contained the captured bacteria, were then streaked onto CHROMagar O157 and Sorbitol MacConkey (SMAC) agar. The DNA extracted from these samples was examined using multiplex PCR to identify potential virulence gene markers, specifically stx-1, stx-2, and eae. Results: Of the 201 examined samples, 88 (43.8%) and 106 (52.7%) were positive for E. coli and produced colorless and mauve colonies on SMAC agar and CHROMagar O157, respectively. Out of 298 isolates in total, 174 isolates of E. coli were isolated with IMB enrichment. The highest detection rate of virulence gene markers was found among isolates that had been isolated using IMB enrichment, where 25 (8.4%), 2 (0.7%) and 12 (4%) isolates tested positive for stx1, stx2, and eae genes respectively. Among 42 isolates harboring potential virulence gene markers, 11 isolates were identified as E. coli O157 (stx₁⁺/eae⁺ or stx₂⁺/eae⁺). ERIC-PCR genotyping was able to determine the genetic relatedness among 42 isolates of E. coli O157 and E. coli non-O157 into 10 types with four identical related clusters and a genetic similarity rate above 90% homology from the identified isolates. Conclusions: The present study gives a clear perspective on STEC contamination in imported frozen beef marketed in Saudi Arabia. Because of the many possibilities of STEC contamination in imported frozen beef, further studies on the spread of STEC at various levels of imported frozen meat are needed on a long-term basis. Full article

Herein, a sensitive biosensor is constructed based on a novel rolling circle amplification (RCA) for colorimetric quantification of lead ion (Pb²⁺). At the detection system, GR5 DNAzymes are modified on the surface of an immunomagnetic bead, and Pb²⁺ is captured by the aptamer, inducing the disintegration of the GR5 DNAzyme and the release of the DNA walker. After the introduction of the template DNA, T4 DNA ligase, and phi29 DNA polymerase, an RCA is initiated for the sensitivity improvement of this method. Moreover, a G4-hemin DNAzyme is formed as a colorimetric signal, owing to its peroxide-like activity to catalyze the TMB-H₂O₂ substrate. Under the optimized conditions, the limit of detection (LOD) of this fabricated biosensor could reach 3.3 pM for Pb²⁺ with a concentration in the range of 0.01–1000 nM. Furthermore, the results of real samples analysis demonstrate its satisfactory accuracy, implying its great potential in the rapid detection of heavy metals in the environment. Full article

Immunomagnetic beads (IMBs) have been widely used to capture and isolate target pathogens from complex food samples. The orientation of the antibody immobilized on the surface of magnetic beads (MBs) is closely related to the effective recognition with an antigen. We put forward an available strategy to orient the antibody on the surface of MBs by changing the charged amino group ratio of the reactive amino groups at optimal pH value. Quantum dots labeling antigen assay, antigen-binding fragment (Fab) accessibility assay and lysine mimicking were used for the first time to skillfully illustrate the antibody orientation mechanism. This revealed that the positively charged ε-NH₂ group of lysine on the Fc relative to the uncharged amino terminus on Fab was preferentially adsorbed on the surface of MBs with a negatively charged group at pH 8.0, resulting in antigen binding sites of antibody fully exposed. This study contributes to the understanding of the antibody orientation on the surface of MBs and the potential application of IMBs in the separation and detection of pathogenic bacteria in food samples. Full article

The problem of rapidly detecting pathogens directly from clinical samples poses significant analytical challenges. Addressing this issue in relation to urinary tract infections, we propose an effective protocol and related immunomagnetic test kits enabling versatile screening for the presence of pathogenic bacteria in unprocessed urine samples. To achieve this, the components of a typical immunomagnetic separation protocol were optimized towards the sensitive assessment of the aggregates formed out of immunomagnetically tagged target pathogens collected from clinical samples. Specifically, a dedicated immunomagnetic material was developed via the functionalization of standardized, micron-sized magnetic beads with generic antibodies against gram-specific bacterial constituents with mannan binding lectin. As such, we demonstrate efficient procedures for achieving the enhanced, specific, and pathogen-mediated cluster formation of these tailored affinity-coated magnetic beads in complex samples. We further show how cluster analysis, in conjunction with the use of nonspecific, inexpensive fluorescent dye, allows for a straightforward optical assessment of the bacterial load directly from urine samples. The optimized sensing protocol and related kits provide, in less than 60 min, qualitative (positive/negative) information on the bacterial load with 85% specificity and 96% sensitivity, which is appropriate to empower clinical microscopy with a new analytic dimension. The procedure is prone to automation, can be conveniently used in clinical microbiology laboratories and, since it preserves the viability of the captured bacteria, can be interfaced with downstream analyses and antimicrobial susceptibility testing. Moreover, the study emphasizes a suite of practical validation assays that are useful for bringing the tool-box of immunomagnetic materials outside the academic laboratory and into real-life applications. Full article

There is an identified need for point-of-care diagnostic systems for detecting and counting specific rare types of circulating cells in blood. By adequately labeling such cells with immunomagnetic beads and quantum dots, they can be efficiently collected magnetically for quantification using fluorescence methods. Automation of this process requires adequate mixing of the labeling materials with blood samples. A static mixing device can be employed to improve cell labeling efficiency and eliminate error-prone laboratory operations. Computational fluid dynamics (CFD) were utilized to simulate the flow of a labeling-materials/blood mixture through a 20-stage in-line static mixer of the interfacial-surface-generator type. Optimal fluid mixing conditions were identified and tested in a magnetic bead/tumor cell model, and it was found that labeled cells could be produced at 1.0 mL/min flow rate and fed directly into an in-line magnetic trap. The trap design consists of a dual flow channel with three bends and a permanent magnet positioned at the outer curve of each bend. The capture of labeled cells in the device was simulated using CFD, finite-element analysis and magnetophoretic mobility distributions of labeled cells. Testing with cultured CRL14777 human melanoma cells labeled with anti-CD146 1.5 μm diameter beads indicated that 90 ± 10% are captured at the first stage, and these cells can be captured when present in whole blood. Both in-line devices were demonstrated to function separately and together as predicted. Full article

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. Full article

In this paper, we developed an isolation system for A549 human lung carcinoma cells as an effective factor for the early diagnosis of lung cancer. A microfluidic immunomagnetic method was used, in which the combination of immunomagnetic separation and a microfluidic system allowed for increased isolation efficiency with uncomplicated manipulation. In the microfluidic immunomagnetic strategy, A549 cells were combined with aptamer-conjugated carboxylated magnetic beads and then collected in a specified region by applying a magnetic field. The results were recorded using a fluorescence microscope, and the captured targets were then quantified. The isolation efficiency of A549 cells is up to 77.8%. This paper developed a simple working procedure, which is less time consuming, high-throughput, and trustworthy for the isolation of A549 cells. This procedure can be a useful reference method for the development of an effective diagnosis and treatment method for lung cancer in the future. Full article

Microbubbles-assisted ultrasound (USMB) has shown promise in improving local drug delivery. The formation of transient membrane pores and endocytosis are reported to be enhanced by USMB, and they contribute to cellular drug uptake. Exocytosis also seems to be linked to endocytosis upon USMB treatment. Based on this rationale, we investigated whether USMB triggers exocytosis resulting in the release of extracellular vesicles (EVs). USMB was performed on a monolayer of head-and-neck cancer cells (FaDu) with clinically approved microbubbles and commonly used ultrasound parameters. At 2, 4, and 24 h, cells and EV-containing conditioned media from USMB and control conditions (untreated cells, cells treated with microbubbles and ultrasound only) were harvested. EVs were measured using flow cytometric immuno-magnetic bead capture assay, immunogold electron microscopy, and western blotting. After USMB, levels of CD9 exposing-EVs significantly increased at 2 and 4 h, whereas levels of CD63 exposing-EVs increased at 2 h. At 24 h, EV levels were comparable to control levels. EVs released after USMB displayed a heterogeneous size distribution profile (30–1200 nm). Typical EV markers CD9, CD63, and alix were enriched in EVs released from USMB-treated FaDu cells. In conclusion, USMB treatment triggers exocytosis leading to the release of EVs from FaDu cells. Full article

Portable detection and quantitation methods for Bacillus anthracis (anthrax) spores in pure culture or in environmental samples are lacking. Here, an amperometric immunoassay has been developed utilizing immunomagnetic separation to capture the spores and remove potential interferents from test samples followed by amperometric measurement on a field-portable instrument. Antibody-conjugated magnetic beads and antibody-conjugated glucose oxidase were used in a sandwich format for the capture and detection of target spores. Glucose oxidase activity of spore pellets was measured indirectly via amperometry by applying a bias voltage after incubation with glucose, horseradish peroxidase, and the electron mediator 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid). Target capture was mediated by polyclonal antisera, whereas monoclonal antibodies were used for signal generation. This strategy maximized sensitivity (500 target spores, 5000 cfu/mL), while also providing a good specificity for Bacillus anthracis spores. Minimal signal deviation occurs in the presence of environmental interferents including soil and modified pH conditions, demonstrating the strengths of immunomagnetic separation. The simultaneous incubation of capture and detection antibodies and rapid substrate development (5 min) result in short sample-to-signal times (less than an hour). With attributes comparable or exceeding that of ELISA and LFDs, amperometry is a low-cost, low-weight, and practical method for detecting anthrax spores in the field. Full article