Immunomagnetic Nanoparticles Research Articles

Innovative approach for mango allergen identification in processed foods using immunomagnetic nanoparticle-enhanced UPLC-MS/MS

Mango (Mangifera indica) is favored for its flavor and nutritional benefits, yet it also poses significant allergenic risks, potentially triggering severe anaphylactic reactions in sensitive individuals. Addressing the urgent need for accurate allergen detection, this study introduces a novel approach utilizing immunomagnetic nanoparticles (IMNPs) combined with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify the 40-kDa major mango allergen in processed foods. By employing 100 nm IMNPs, the method effectively isolates and concentrates the allergens from complex food matrices, facilitating precise UPLC-MS/MS analysis. Demonstrating exceptional specificity, the technique accurately distinguishes mango allergens from those in nine other fruits and vegetables. It achieves low limits of detection (LODs) of 0.032 μg/mL in juice and 0.165 μg/g in cookies. When tested on 34 commercial products and compared with a real-time PCR method approved by the Taiwan FDA, the method validated its high specificity (100%) and sensitivity (92.9%), based on the ingredient labeling of these products. These advancements mark a significant leap in allergen detection technology, offering a sensitive, specific, and practical solution for identifying mango allergen residues in processed foods, thereby enhancing food safety protocols and protecting public health.

Preparation of immunomagnetic composite nanostructures with bifunctional four-arm PEG derivatives as linkers for the ultrafast enrichment of zearalenone and its metabolites

It is challenging to prepare sample pretreatment materials with simple use, strong selectivity and satisfactory enrichment performance. In this study, the antibody (3D4) that can specifically recognize zearalenone (ZEN) and its metabolites was immobilized on the surface of gold-coated magnetic Fe3O4 nanoparticles (GMN) by streptavidin (SA)-biotin interaction using GMN as the substrate and our designed four-arm PEG derivative (HS-4ARMPEG10K-(CM)3) as the linker. The immunomagnetic nanoparticles (GMN-4ARMPEG10K-SA-3D4) prepared by this strategy can achieve rapid enrichment (only 5 min) of analytes directly in the matrix, and higher enrichment capacity compared with the previous immunomagnetic particles. The sensitive and accurate analysis of ZEN and its metabolites can be achieved coupled with HPLC-MS/MS. The LODs and LOQs were 0.02–0.05 μg/kg and 0.05–0.10 μg/kg, respectively. The recoveries were 84.13%–112.67%, and the RSDs were 1.09%–9.39%. The method can provide a powerful tool for highly sensitive and rapid monitoring of mycotoxins in complex matrices due to its’ strong selectivity and resistance to matrix interference.

Rapid Homogeneous Fluorescent Enzyme-Linked Immunosorbent Assay (ELISA) for N-Terminal Brain Natriuretic Peptide (NT-proBNP) Combining Magnetic Probes and Ampliflu Red

Heart failure (HF) is the leading cause of death from cardiovascular disease, and early diagnosis using serum markers holds extraordinary significance for the treatment of patients. N-terminal brain natriuretic peptide (NT-proBNP) is the gold standard for HF diagnosis. Herein, a novel, rapid, sensitive, and immune-sensing strategy for NT-proBNP is reported employing an enzyme-linked immunosorbent assay (ELISA) that combines the use of magnetic biological probes and the fluorogenic substrate Ampliflu Red. Functionalized immunomagnetic nanoparticles with large surface areas provide an abundant reaction platform and reduce the analysis time due to their superparamagnetic properties. Ampliflu Red is catalyzed by horseradish peroxidase (HRP) using hydrogen peroxide (H2O2) to produce strong fluorescence and a low background. The reported method achieved a good detection limit of 2.73 ng mL−1 and a wide linear range from 3.13 to 100 ng mL−1. The analysis time was less than 1 h, which is superior to that of traditional ELISA (3–4 h). In addition, the recoveries of spiked human serum were satisfactory, indicating that the proposed strategy has potential applications for the rapid determination of NT-proBNP and other biomarkers in clinical settings.

Magnetic properties and electron oxidation state transition of immunomagnetic nanoparticles specifically captured with the target bacteria

Immunomagnetic nanoparticles (IMNPs) have been widely applied for the capture and concentration in the rapid detection of target bacteria. In this research, the focus was on studying the changes in magnetic properties changes of the IMNPs when they were attached to bacterial cells. These alterations in properties could facilitate an even rapid detection of the target bacteria and eliminate the need for culturing on plating media. The variation of magnetizing values, including saturated magnetization (Ms), remanent magnetization (Mr), coercivity force (Hc), and magnetic susceptibility (χm), was analysed through M-H loops. It was observed that the magnetizing properties of the IMNPs underwent changes based on the concentrations of Salmonella Typhimurium cells in the test solution. The correlation of this phenomenon was confirmed by the results of synchrotron x-ray absorption spectroscopy (XAS), which revealed electronic transition changes in the IMNPs after capturing the bacteria cells. Additionally, the electronic bands of the magnetite nanoparticle [Fe(II) and Fe(III)] were detected, indicating an electronic transformation between the Salmonella cells and the bound IMNPs. The XAS change was further verified using different cell types, such as Campylobacter jejuni which also showed electronic transformation after attaching to IMNPs. These findings suggest that IMNP-cell attachment triggered the change in the magnetic properties of IMNPs. Such insights could serve as valuable information for the development of novel rapid bacteria detection assays/devices using magnetic sensing techniques.

Genetically Engineered Cell Membrane‐Coated Magnetic Nanoparticles for High‐Performance Isolation of Circulating Tumor Cells

AbstractCirculating tumor cells (CTCs) are a crucial biomarker for early cancer diagnosis and progress of cancer metastasis. However, the extremely rare CTCs with large amounts of background leukocytes seriously restricte the purity of enriched CTCs. Herein, genetically engineered cell membrane‐coated magnetic nanoparticles with following properties are constructed: I) The leukocyte membrane camouflaged nanoparticles, which could significantly reduce nonspecific binding of homologous leukocytes and achieve high‐purity isolation of CTCs, are prepared by simple one‐step extrusion and II) the cellular membrane stably expressing single‐chain variable fragment (scFv) of anti‐epidermal growth factor receptor (EGFR) antibody could ensure the recognition of EGFR‐positive CTCs. The binding affinity of the resulting nanoparticles toward extracellular EGFR protein improved more than 100‐fold compared with natural cell membrane‐coated nanoparticles. Furthermore, compared with commercial immunomagnetic nanoparticles, the functional nanoparticles achieved a greater capture efficiency in artificial blood samples and the cell purity increased from 64.8% to 93.5%. Consequently, the authors successfully isolate high pure CTCs from 6 out of 6 cancer patients using engineered cell membrane‐coated nanoparticles. This platform exhibits a promising potential for CTC detection in clinical samples, offering an innovative method for cancer diagnosis and prognosis evaluation.

Impact of immunomagnetic nanoparticle size on capture efficiency, bioactivity, and proliferation ability of circulating tumor cells

Impact of immunomagnetic nanoparticle size on capture efficiency, bioactivity, and proliferation ability of circulating tumor cells

Dual Stealth Functional Immuno-magnetic Nanoparticles for High-Performance Isolation of Circulating Tumor Cells.

As a biomarker of hepatocellular carcinoma (HCC) biopsy, circulating tumor cells (CTCs) are often used in the diagnosis of cancer and treatment guidance. For CTCs detection, immuno-magnetic nanoparticles (IMNs) are one of the most commonly used platforms. However, the nonspecific adsorption of proteins and non-tumor cells weakens the performance of IMNs to capture CTCs. In this work, we developed an IMNs platform which was constructed by a biomimetic protein corona precoating and a polyethylene glycol (PEG) spacer to form the PEG and corona-coated IMNs (IP-CMNs). Due to the dual stealth effect of protein corona precoating and PEG spacer, the nonspecific protein adsorption and cell binding of P-CMNs could reduce by ∼5.5- and ∼5.4-fold, respectively, compared with those of unmodified particles. Furthermore, the PEG spacer could not only reduce the interaction between IP-CMNs and leukocytes but also enhance the capture performance toward tumor cells. By using artificial blood samples, the capture efficiency of IP-CMNs toward rare CTCs was found to be 88.3%, while it was 70.5% by using commercial IMNs. Finally, CTCs were successfully isolated in all HCC patient blood samples (7/7) using IP-CMNs. These results provide insight into the use of the multifunctional nanoplatform as a useful tool for CTCs detection.

Multiple electromagnet synergistic control enabled fast and automatic biosensing of Salmonella in a sealed microfluidic chip

Point-of-care testing of pathogens is vital for prevention of food poisoning. Herein, a colorimetric biosensor was elaborately developed to rapidly and automatically detect Salmonella in a sealed microfluidic chip with one central chamber for housing immunomagnetic nanoparticles (IMNPs), bacterial sample and immune manganese dioxide nanoclusters (IMONCs), four functional chambers for housing absorbent pad, deionized water and H2O2-TMB substrate, and four symmetric peripheral chambers for achieving fluidic control. Four electromagnets were placed under peripheral chambers and synergistically controlled to manipulate their respective iron cylinders at the top of these chambers for deforming these chambers, resulting in precise fluidic control with designated flowrate, volume, direction and time. First, the electromagnets were automatically controlled to mix IMNPs, target bacteria and IMONCs, resulting in the formation of IMNP-bacteria-IMONC conjugates. Then, these conjugates were magnetically separated by a central electromagnet and the supernatant was directionally transferred to the absorbent pad. After these conjugates were washed by deionized water, the H2O2-TMB substrate was directionally transferred to resuspend the conjugates and catalyzed by the IMONCs with peroxidase-mimic activity. Finally, the catalysate was directionally transferred back to its initial chamber, and its color was analyzed by the smartphone APP to determinate bacterial concentration. This biosensor could detect Salmonella quantitatively and automatically in 30 min with a low detection limit of 101 CFU/mL. More importantly, the whole bacterial detection procedure from bacterial separation to result analysis was achieved on a sealed microfluidic chip through multiple electromagnet synergistic control, and this biosensor has great potential for point-of-care testing of pathogens without cross contaminations.

Monte Carlo simulation of micro-x-ray fluorescence (μ-XRF) based detection of immunomagnetically labeled tumor cells

Objective. Circulating tumor cells (CTCs) carry crucial information related to the spreading and proliferation of tumors, especially at early stages of the disease. Despite the huge clinical potential held by CTCs in cancer therapy, capture and detection of these cells from the patient's peripheral blood system is rather challenging since CTCs are extremely rare cells. The objective of this paper is, based on Monte Carlo simulations, to propose the detection of immunomagnetically labelled tumor cells by micro-x-ray fluorescence (μ-XRF). Approach. The simulations were carried out with the Monte Carlo N-Particle, version 6.2, (MCNP6.2) code. The model simulates 20 μm cancer cell lines and 10 μm CTCs tagged with Fe3O4@SiO2 spherical nanoparticles of diameters 25 nm, 60 nm and 110 nm. A 17.5 keV monochromatic, micro-focused x-ray beam of diameter 15 μm, impinges on cancer cells immersed in a phosphate-buffered saline solution. The simulations also include a polymeric sample holder and a silicon drift detector with a beryllium window and silver collimator. Main results. The results show the dependence of the signal intensity (Fe Kα line) on cell and nanoparticle sizes. Samples containing two and three CTCs were also simulated in particular geometrical configurations. It is presented how the inter-cell distances and cell positions relative to the incident x-ray beam affect the signal. In addition, within the parameters used in the simulations, μ-XRF method provides a minimum detection limit of 9.4 pg of Fe, which corresponds to detecting a single 10 μm CTC labeled with 110 nm Fe3O4@SiO2 nanoparticles at 6.3% binding. Significance. The μ-XRF based method proposed in this paper for detecting CTCs, combined with immunomagnetic nanoparticles (NPs), has the potential to be innovative in the field of liquid biopsy.

Self-assembled supramolecular immunomagnetic nanoparticles through π-π stacking strategy for the enrichment of circulating tumor cells.

Owing to their high-specific binding toward targets as well as fast and convenient separation operations, immunomagnetic beads (IMBs) are widely used in the capture and detection of circulating tumor cells (CTCs). To construct the IMBs, surface modifications are generally performed to functionalize the magnetic cores (e.g. Fe3O4 nanoparticles), and the employed surface modification strategies normally influence the structure and functions of the prepared IMBs in return. Different from the existing work, we proposed the use of supramolecular layer-by-layer (LBL) self-assembly strategy to construct the IMBs. In general, owing to the π-π stacking interactions, the polydopamine, graphene oxide and 'molecular glue' γ-oxo-1-pyrenebutyric acid were self-assembled on Fe3O4 nanoparticles sequentially, thereby accomplishing the integration of different functional components onto magnetic cores to prepare the self-assembled supramolecular immunomagnetic beads (ASIMBs). The ASIMBs showed high sensitivity, specificity and good biocompatibility to the model CTCs and low nonspecific adsorption to the negative cells (∼93% for MCF-7 cells and 17% for Jurkat cells). Meanwhile, ASIMBs possessed a remarkable potential to screen the rare MCF-7 cells out of large amounts of interfering Jurkat cells with the capture efficiency of 75-100% or out of mouse whole blood with the capture efficiency of 20-90%. The captured cells can be further recultured directly without any more treatment, which showed huge applicability of the ASIMBs for in vitro detection in clinical practices.

Abstract PR007: Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients

Abstract Background: Genomic profiling has revolutionized precision oncology impacting the diagnosis, prognosis, and therapy decisions. Considering high spatiotemporal diversity and heterogenicity of breast tumor-cell genomes, small-gene panels often fail to capture rare but important genomic alterations. Conversely, comprehensive ctDNA sequencing approaches enable the identification of under characterized ‘long tailed driver’ genomic alterations and capture Intra and inter metastatic heterogeneity. Here, we demonstrate the clinical utility of comprehensive genome profiling with higher sensitivity to predict the possibility of metastasis in early-stage breast cancer patients. Methods: We retrospectively analyzed ctDNA and genomic DNA (gDNA) from FFPE samples as well as circulating tumor cells (CTC) in 10 treatment-naive hormone positive and HER2 negative, primary-stage breast cancer patients [GS1] using the OncoIndx comprehensive 600 gene panel. The panel captures all important cancer-relevant genomic alterations including Tumor Mutation Burden (TMB), Micro Satellite Instability (MSI), homologous recombination deficiency (HRD) prediction, and cfDNA tumor fraction (TF). CTCs were enumerated from 1.5 ml of blood using the OncoDiscover platform approved by the Drug Controller General of India having anti-EpCAM antibody-mediated immunomagnetic nanoparticles. CTCs were confirmed for cytokeratin 18+ and DAPI + markers and the absence of CD45. Results: Comprehensive genomic profile obtained from ctDNA and gDNA from FFPE of early-stage breast cancer patients predominantly exhibited the presence of alterations in PIK3CA and ESR1 signaling pathways. PIK3CA mutations were present in 77% and 44% of baseline ctDNA and gDNA samples, while ESR1 mutations were present in 44% and 22% of baseline ctDNA and gDNA, respectively. In addition, we observed about 70% additional driver mutations in ctDNA samples suggesting shedding of ctDNA together with CTC (80% positive), a likely positive biomarker of metastasis. About 50% of the patients showed higher TMB and HRD. Notably, TF representing ctDNA varied between 13% to 27% in blood samples with a corresponding ploidy range of 2.9 to 4.7. Surprisingly, ~50% of the patient population matched the mutation profile of clinically confirmed metastatic patients. All the patients harboring potential metastatic driver alterations showed the presence of CTCs in peripheral blood. Conclusions: Comprehensive ctDNA genomic profiling showed potential metastasis driving alterations suggesting the role of ctDNA-based liquid biopsy to predict metastasis in early breast cancer patients. We observed enhanced TF at the time of diagnosis, possibly due to the presence of distant metastasis, high disease burden, and aggressive tumor biology. Our results suggest that ctDNA dynamics at the time of disease presentation can predict early metastasis, and may demonstrate the divergent response of tumor heterogeneity to treatment in early-stage breast cancer. Citation Format: Gowhar Shafi, Manoj Dongare, Atul Bharde, Moubeen Fauzul, Kanchan Hariramani, Alain D’Souza, Bhagwat Jadhav, Trupti Kad, Sangeeta Prajapati, Vikas Jadhav, ManojKumar Kumaran, Sumit Haldar, Vatsal Mehra, Sujit Joshi, Gourishankar Aland, Richa Dave, Sreeja Jayant, Aravindan Vasudevan, Mohan Uttarwar, Jayant Khandare. Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR007.

Magnetorheological elastomer and smartphone enable microfluidic biosensing of foodborne pathogen

Rapid detection of foodborne pathogens is crucial to prevent the outbreaks of foodborne diseases. In this work, we proposed a novel microfluidic biosensor based on magnetorheological elastomer (MRE) and smartphone. First, micropump and microvalves were constructed by deforming the MRE under magnetic actuation and integrated into the microfluidic biosensor for fluidic control. Then, the micropump was used to deliver immune porous gold@platinum nanocatalysts (Au@PtNCs), bacterial sample, and immunomagnetic nanoparticles (MNPs) into a micromixer, where they were mixed, incubated and magnetically separated to obtain the Au@PtNC-bacteria-MNP complexes. After 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide were injected and catalyzed by the Au@PtNCs, smartphone was used to measure the color of the catalysate for quantitative analysis of target bacteria. Under optimal conditions, this biosensor could detect Salmonella typhimurium quantitatively and automatically in 1 h with a linear detection range of 8.0 × 101 CFU/mL to 8.0 × 104 CFU/mL and a detection limit of 62 CFU/mL. The microfluidic biosensor was compact in size, simple to use, and efficient for detection, and might be used for in-field screening of foodborne pathogens to prevent food poisoning.

Ultrasensitive plasmonic photothermal immunomagnetic bioassay using real-time and end-point dual-readout

One of the most straightforward and sensitive methods for combining the specificity of immunological recognition with the amplification capability of polymerase chain reaction (PCR) is the fluorescence-based immuno-PCR. But up until now, the majority of PCR-based immunoassays have utilized the pricey and lengthy Peltier-based heating technique. Herein, we introduce a plasmonic photothermal quantitative immuno-PCR (PPT-qiPCR) assay with real-time and end-point dual readout for biomarker detection using multifunctional plasmonic immunomagnetic nanoparticles (PIMNs). PPT-qiPCR is simple, time-saving and inexpensive compared with conventional iPCR platforms using PCR machine. We showed that an immunomagnetic technique utilizing PIMNs can shorten the time needed for collection, washing, and concentrating target proteins in the enzyme-linked immunosorbent assay (ELISA) procedure. Real-time readout also permits quick preliminary discrimination of target proteins with apparent sensitivity in commercial ELISA investigations. Additionally, in-situ end-point fluorescence or colorimetric detection using only the human eye considerably improve the assay sensitivity. Our PPT-qiPCR can significantly advance next-generation point-of-care molecular diagnostic technology and is anticipated to be immediately useful for the sensitive and quick identification of several disease biomarkers.

Novel aerosol detection platform for SARS‑CoV‑2: Based on specific magnetic nanoparticles adsorption sampling and digital droplet PCR detection

The SARS‑CoV‑2 virus is released from an infectious source (such as a sick person) and adsorbed on aerosols, which can form pathogenic microorganism aerosols, which can affect human health through airborne transmission. Efficient sampling and accurate detection of microorganisms in aerosols are the premise and basis for studying their properties and evaluating their hazard. In this study, we built a set of sub-micron aerosol detection platform, and carried out a simulation experiment on the SARS‑CoV‑2 aerosol in the air by wet-wall cyclone combined with immunomagnetic nanoparticle adsorption sampling and ddPCR. The feasibility of the system in aerosol detection was verified, and the influencing factors in the detection process were experimentally tested. As a result, the sampling efficiency was 29.77%, and extraction efficiency was 98.57%. The minimum detection limit per unit volume of aerosols was 250 copies (102 copies/mL, concentration factor 2.5).

Power-free microfluidic biosensing of Salmonella with slide multivalve and disposable syringe

In this study, a power-free biosensor was presented to detect Salmonella typhimurium on a microfluidic chip using a slide multivalve for channel selection and a disposable syringe for fluidic transfer. First, bacterial sample with immunomagnetic nanoparticles (IMNPs) and glucose oxidase (GOx) modified immune polystyrene nanoparticles (IPNPs), washing buffer, glucose, and peroxide test strip (PTS) were preloaded in their respective chambers at the periphery of chip. After the slide multivalve was selected to connect sample chamber with common separation chamber, which was connected with a syringe, the mixture of Salmonella, IMNPs and IPNPs was back and forth moved through 3D Tesla-structure micromixer using the syringe, resulting in the formation of IMNP-Salmonella-IPNP complexes, which were captured in the separation chamber using a magnet. Then, two washing chambers were selectively connected respectively to remove sample background and excessive IPNPs, and glucose chamber was connected, allowing the GOx to catalyze glucose to produce hydrogen peroxide in the separation chamber. Finally, PTS chamber was connected and the catalysate was transferred from the separation chamber to the PTS chamber, leading to the color change of PTS, followed by using smartphone App to collect and analyze the image of PTS for bacterial determination. The simple biosensor enabled simple detection of Salmonella as few as 130 CFU/mL within 60 min and is promising for practical applications in the resource-limited regions due to its low cost, simple operation, and small size.

Neutrophil membrane-coated immunomagnetic nanoparticles for efficient isolation and analysis of circulating tumor cells

The isolation and analysis of scarce circulating tumor cells (CTCs) with immunomagnetic nanoparticles (IMNs) have shown promising outcomes in noninvasive cancer diagnosis. However, the IMNs adsorb nonspecific proteins after entering into biofluids and the formed protein coronas cover surface targeting ligands, limiting the detection efficiency of IMNs. In addition, the interaction between surface targeting ligands and white blood cells (WBCs) significantly limits the purity of CTCs isolated by IMNs. Furthermore, the interfacial collision of nanoparticles and cells has negative effects on the viability of isolated CTCs. All of these limitations synthetically restrict the isolation and analysis of rare CTCs for early diagnosis and precision medicine. Here, we proposed that surface functionalization of IMNs with neutrophil membranes can simultaneously reduce nonspecific protein adsorption, enhance the interaction with CTCs, reduce the distraction from WBCs, and improve the viability of isolated CTCs. In spiked blood samples, our neutrophil membrane-coated IMNs (Neu-IMNs) exhibited a superior separation efficiency from 41.36% to 96.82% and an improved purity from 40.25% to 90.68% when compared to bare IMNs. Additionally, we successfully isolated CTCs in 19 out of total 20 blood samples from breast cancer patients using Neu-IMNs and further confirmed the feasibility of the isolated CTCs for downstream cell sequencing. Our work provides a new perspective on engineered IMNs for efficient isolation and analysis of CTCs, paving the way for early noninvasive diagnosis of cancer.

Hyperspectral dark-field microscopy for pathogen detection based on spectral angle mapping

Early screening for food pathogens, specifically Escherichia coli O157:H7 in ready-to-eat food remains a central challenge, hyperspectral imaging (HSI) has the potential to acquire both image and spectral information of the target and is simple enough to serve as an effective auxiliary technology in food monitoring. Herein, a visual method was developed based on spectral angle mapping (SAM) and dark-field microscopy for rapid detection and quantification of Escherichia coli. First, immunomagnetic nanoparticle (MNP-probe) and gold nanoparticle (AuNP-probe) probes were prepared to form a sandwich complex for efficient concentration of the target bacteria. The complexes were then dropped directly on a glass slide for colony counting with hyperspectral dark field microscopy. The AuNP-probe bound to the complex was counted and SAM was used to evaluate the spectra and HSI image data to distinguish the target pixel from the background. The linear detection range of the method was estimated to be in the range between 6.1 × 101 and 6.1 × 106 CFU/mL in the milk sample, and the detection limit (LOD) of this method was estimated to be ~61 CFU/mL. The average recovery of organisms in the milk sample was 106.0%, indicating that this method can be utilized to detect Escherichia coli in food samples and serve as an effective method to monitor pathogens in a complex sample.

Dual-Aptamer-Targeted Immunomagnetic Nanoparticles to Accurately Explore the Correlations between Circulating Tumor Cells and Gastric Cancer.

It has been acknowledged that circulating tumor cells (CTCs) are promising biomarkers in liquid biopsy for cancer diagnosis and prognosis. However, the relationship between the CTC number and gastric cancer has scarcely been quantitatively investigated. Moreover, the single criterion of epithelial cell adhesion molecule (EpCAM) antibody/aptamer to specifically recognize epithelial CTCs cannot be universally applied for clinical applications, as it fails to recognize EpCAM-negative CTCs. Herein, we propose simple, low-cost, dual-aptamer (EpCAM and PTK7)-modified immunomagnetic Fe3O4 particles (IMNs) for efficient capture of heterogeneous CTCs and downstream analysis in gastric cancer patients. High PTK7 expression and a significant negative correlation between PTK7 and EpCAM expression were observed in primary gastric cancer tissues. Taking MGC-803 and BGC-823 cells as CTC models, the obtained dual-targeting IMNs could distinguishably recognize these cells with both high or low EpCAM and PTK7 expressions, which enhanced the accuracy of CTC recognition in gastric cancer. More than 95% of these two kinds of cells could be captured within 20 min of incubation, which was significantly more efficient than that of single EpCAM- or PTK7-modified IMNs. With this strategy, as low as five CTCs could be captured from phosphate-buffered saline (PBS), a cell mixture containing THP-1 cells, and lysed blood mediums. Moreover, the obtained CTCs can be used for subsequent gene analysis. Finally, the fabricated IMNs were successfully applied for CTC capture in 1.0 mL of peripheral blood samples from patients with gastric cancer. The detected CTC numbers in 72 participants were found to have close relationships with chemotherapy sensitivity, diagnosis, stage, and distant metastasis of patients. This work provides important references for further investigations on CTC-related diagnosis and individualized treatment.

Artificial cell membrane camouflaged immunomagnetic nanoparticles for enhanced circulating tumor cell isolation.

Precise and specific circulating tumor cell (CTC) isolation is heavily interfered with by blood cells and proteins. Although satisfactory results have been achieved by some cell membrane-derived platforms, the following limitations have seriously limited the commercialization potential: complex membrane composition, difficult batch difference control, inconvenient source cell expansion, etc. To overcome these limitations, artificial cell membrane camouflage made from commercialized lipids and proteins was proposed in this work. Specifically, a biotinylated phospholipid which can serve as a lipid component and provide active sites (biotin) for antibody modification, and human serum albumin (HSA), which can effectively reduce certain blood protein adsorption, were applied simultaneously to endow our immunomagnetic platform with a new biological identity. Besides, making full use of the robust lipid and protein absorption ability of graphene nanosheets (GNs), the biomimetic cell membrane can be easily integrated into the magnetic core through simple lipid and protein solution incubation. Surprisingly, the resulting artificial cell membrane camouflaged immunomagnetic nanoparticles (AIMNPs) achieved high specificity (average capture efficiency 87.0%), good sensitivity (7 model CTCs per 0.5 mL) and an enhanced anti-nonspecific absorption ability (15-105 white blood cells per 1.5 mL) in both mimic and clinical blood samples.

Protein corona-coated immunomagnetic nanoparticles with enhanced isolation of circulating tumor cells.

Immunomagnetic nanoparticles (IMNs) have been widely developed as a detection tool to isolate rare circulating tumor cells (CTCs) from whole blood as a potential method for early cancer diagnosis, metastasis examination, and treatment guidance. However, a spontaneous interaction between nanoparticles and proteins results in the formation of a protein corona that reduces the performance of IMNs when they enter body fluids. To address this issue, the protein corona was precoated onto magnetic nanoparticles (C-MNs), and then their surfaces were conjugated with an immuno-antibody. The adsorption of proteins on C-MNs was decreased 6-fold and non-specific cell binding was reduced 5-fold, compared with magnetic nanoparticles (MNs). Furthermore, the immuno-antibody functionalized C-MNs (IC-MNs) maintained highly specific CTC capture performance when exposed to blood plasma. By using artificial spiked blood samples, IC-MNs exhibited 90.2% CTC isolation efficiency, compared with 60.3% by using IMNs. IC-MNs also successfully captured CTCs with high purity in 24 out of 26 female breast cancer patient blood samples. This work demonstrated that a novel preformed protein corona strategy can provide a useful clinically applicable diagnostic tool.