Analysis of maternal Fc gamma receptor IIIb isoantibodies using immunomagnetic negative selected neutrophils.

BACKGROUND AND OBJECTIVES: The isolation of neutrophils and subsequent detection of anti-human neutrophil antigens (HNA) antibodies are crucial in clinical medicine for the diagnosis of autoimmune neutropenia, neonatal alloimmune neutropenia (NAIN) and transfusion-related acute lung injury (TRALI). This study reports two cases of maternal anti-Fc-gamma-receptor-IIIb (FcγRIIIb) isoimmunization without NAIN symptoms and compares the efficiency of immunomagnetic negative selection (IMNS) with traditional dextran/Ficoll for neutrophil isolation in HNA serological assays. MATERIALS AND METHODS: Investigating two cases of maternal anti-FcγRIIIb isoimmunization, neutrophils from three donors were isolated from 8 mL of whole blood using IMNS and dextran/Ficoll. Serological assays included the granulocyte agglutination and immunofluorescence test, monoclonal antibody immobilization of granulocyte antigens and the LABScreen Multi (One Lambda). IMNS and dextran/Ficoll were compared in terms of cell yield, viability, time, cost and purity. RESULTS: Maternal anti-FcγRIIIb isoantibodies with FCGR3B gene deletion were detected in both cases. Newborns and fathers exhibited specific gene combinations: FCGR3B*02/FCGR3B*02 (Case 1) and FCGR3B*02/FCGR3B*03 (Case 2). IMNS outperformed dextran/Ficoll, yielding four times more neutrophils (average neutrophil counts: 18.5 × 103/µL vs. 4.5 × 103/µL), efficiently removing non-neutrophil cells and reducing processing time (30-40 min vs. 70-90 min), although it incurred a higher cost (2.7 times). CONCLUSION: Two cases of maternal anti-FcγRIIIb isoantibodies, unrelated to NAIN, were identified. Although neutropenia has not been described in these cases, we emphasize the importance of identifying asymptomatic cases with the potential for severe neutropenia. Additionally, IMNS is introduced as a rapid, high-yield, high-purity neutrophil isolation technique, beneficial for serological assays detecting anti-HNA antibodies.

Enhancing the sensitivity of immunomagnetic assay for 3-phenoxybenzonic acid: a novel approach utilizing magnetosome-nanobody complexes and gold nanoparticles probes.

The 3-phenoxybenzoic acid (3-PBA) residues in environment are posing a significant challenge to our daily lives. To establish a more sensitive and rapid detection method, anti-3-PBA nanobodies (Nbs) were immobilized onto magnetosomes (bacterial magnetic nanoparticles, termed as BMPs), forming a robust BMP-Nb complex. The 3-PBA derivative was labeled with horseradish peroxidase (HRP) and further associated with gold nanoparticles (AuNPs) to create a highly sensitive probe (3-PBA-HRP-AuNP). An innovative immunoassay that combined BMP-Nb complex with 3-PBA-HRP-AuNP was developed for determinaton of 3-PBA. This method enabled the determination of 3-PBA with a half-maximum signal inhibition concentration (IC50) of 1.03 ng/mL, which was more sensitive than that of using 3-PBA-HRP as tracer with an IC50 of 2.18 ng/mL. The reliability of the assay was evidenced by the quantitative recovery of 3-PBA from water and soil samples ranging from 76.85 to 95.64%. The 3-PBA residues determined by this assay in actual water samples were between < LOD and 2.54 ng/mL and were between < LOD and 11.25 ng/g (dw) in real soils, respectively, which agreed well with those of liquid chromatography mass spectrometry (LC-MS). Collectively, the BMP-Nb and 3-PBA-HRP-AuNP-based immunoassay provides a powerful tool for the precise detection of 3-PBA residues in environment matrices, reinforcing our capacity to monitor and mitigate potential ecological and health impacts associated with this prevalent pollutant.

Fluorescent identification of immunomagnetically captured CTCs using triplex-aptamer-targeted dendritic SiO2@Fe3O4 nanocomposite.

The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of cancer patients. There is a compelling need to develop an efficient technique for the capture and identification of these rare CTCs. However, the exclusive reliance on a single criterion, such as the epithelial cell adhesion molecule (EpCAM) antibody or aptamer, for the specific recognition of epithelial CTCs is not universally suitable for clinical applications, as it usually falls short in identifying EpCAM-negative CTCs. To address this limitation, we propose a straightforward and cost-effective method involving triplex fluorescently labelled aptamers (FAM-EpCAM, Cy5-PTK7, and Texas Red-CSV) to modify Fe3O4-loaded dendritic SiO2 nanocomposite (dmSiO2@Fe3O4/Apt). This multi-recognition-based strategy not only enhanced the efficiency in capturing heterogeneous CTCs, but also facilitated the rapid and accurate identification of CTCs. The capture efficiency of heterogenous CTCs reached up to 93.33%, with a detection limit as low as 5 cells/mL. Notably, the developed dmSiO2@Fe3O4/Apt nanoprobe enabled the swift identification of captured cells in just 30 min, relying solely on the fluorescently modified aptamers, which reduced the identification time by approximately 90% compared with the conventional immunocytochemistry (ICC) technique. Finally, these nanoprobe characteristics were validated using blood samples from patients with various types of cancers.

An integrated magnetic separation enzyme-linked colorimetric sensing platform for field detection of Escherichia coli O157: H7 in food.

An intelligent colorimetric sensing platform integrated with in situ immunomagnetic separation function was developed for ultrasensitive detection of Escherichia coli O157: H7 (E. coli O157: H7) in food. Captured antibody modified magnetic nanoparticles (cMNPs) and detection antibody/horseradish peroxidase (HRP) co-functionalized AuNPs (dHAuNPs) were firstly synthesized for targeted enrichment and colorimetric assay of E. coli O157: H7, in which remarkable signal amplification was realized by loading large amounts of HRP on the surface of AuNPs. Coupling with the optical collimation attachments and embedded magnetic separation module, a highly integrated optical device was constructed, by which in situ magnetic separation and high-quality imaging of 96-well microplates containing E. coli O157: H7 was achieved with a smartphone. The concentration of E. coli O157: H7 could be achieved in one-step by performing digital image colorimetric analysis of the obtained image with a custom-designed app. This biosensor possesses high sensitivity (1.63 CFU/mL), short detecting time (3 h), and good anti-interference performance even in real-sample testing. Overall, the developed method is expected to be a novel field detection platform for foodborne pathogens in water and food as well as for the diagnosis of infections due to its portability, ease of operation, and high feasibility.

Combining rVAR2 and Anti-EpCAM to Increase the Capture Efficiency of Non-Small-Cell Lung Cancer Cell Lines in the Flow Enrichment Target Capture Halbach (FETCH) Magnetic Separation System.

Circulating tumor cells (CTCs) are detected in approximately 30% of metastatic non-small-cell lung cancer (NSCLC) cases using the CellSearch system, which relies on EpCAM immunomagnetic enrichment and Cytokeratin detection. This study evaluated the effectiveness of immunomagnetic enrichment targeting oncofetal chondroitin sulfate (ofCS) using recombinant VAR2CSA proteins (rVAR2) to improve the recovery of different NSCLC cell lines spiked into lysed blood samples. Four NSCLC cell lines-NCI-H1563, A549, NCI-H1792, and NCI-H661-were used to assess capture efficiency. The results demonstrated that the combined use of anti-EpCAM antibody and rVAR2 significantly enhanced the capture efficiency to an average of 88.2% compared with 40.6% when using only anti-EpCAM and 56.6% when using only rVAR2. These findings suggest that a dual-marker approach using anti-EpCAM and rVAR2 can provide a more robust and sensitive method for CTC enrichment in NSCLC, potentially leading to better diagnostic and prognostic outcomes.

Boosting SARS-CoV-2 Enrichment with Ultrasmall Immunomagnetic Beads Featuring Superior Magnetic Moment.

The isolation and enrichment efficiency of SARS-CoV-2 virus in complex biological environments is often relatively low, presenting challenges in direct detection and an increased risk of false negatives, particularly during the early stages of infection. To address this issue, we have developed a novel approach using ultrasmall magnetosome-like nanoparticles (≤10 nm) synthesized via biomimetic mineralization of the Mms6 protein derived from magnetotactic bacteria. These nanoparticles are surface-functionalized with hydrophilic carboxylated polyethylene glycol (mPEG2000-COOH) to enhance water solubility and monodispersity. Subsequently, they are coupled with antibodies targeting the receptor-binding domain (RBD) of the virus. The resulting magnetosome-like immunomagnetic beads (Mal-IMBs) exhibit high magnetic responsiveness comparable to commercial magnetic beads, with a saturation magnetization of 90.6 emu/g. Moreover, their smaller particle size provides a significant advantage by offering a higher specific surface area, allowing for a greater number of RBD single-chain fragment variable (RBD-scFv) antibodies to be coupled, thereby enhancing immune capture ability and efficiency. To validate the practicality of Mal-IMBs, we evaluated their performance in recognizing the RBD antigens, achieving a maximum capture ability of 83 µg/mg per unit mass. Furthermore, we demonstrated the binding capability of Mal-IMBs to SARS-CoV-2 pseudovirus using fluorescence microscopy. The Mal-IMBs effectively enriched the pseudovirus at a low copy concentration of 70 copies/mL. Overall, the small Mal-IMB exhibited excellent magnetic responsiveness and binding efficiency. By employing a multisite virus binding mechanism, it significantly improves the enrichment and separation of SARS-CoV-2 in complex environments, facilitating rapid detection of COVID-19 and contributing to effective measures against its spread.

Microfluidic-Assisted CTC Isolation and In Situ Monitoring Using Smart Magnetic Microgels.

Capturing rare disease-associated biomarkers from body fluids can offer an early-stage diagnosis of different cancers. Circulating tumor cells (CTCs) are one of the major cancer biomarkers that provide insightful information about the cancer metastasis prognosis and disease progression. The most common clinical solutions for quantifying CTCs rely on the immunomagnetic separation of cells in whole blood. Microfluidic systems that perform magnetic particle separation have reported promising outcomes in this context, however, most of them suffer from limited efficiency due to the low magnetic force generated which is insufficient to trap cells in a defined position within microchannels. In this work, a novel method for making soft micromagnet patterns with optimized geometry and magnetic material is introduced. This technology is integrated into a bilayer microfluidic chip to localize an external magnetic field, consequently enhancing the capture efficiency (CE) of cancer cells labeled with the magnetic nano/hybrid microgels that are developed in the previous work. A combined numerical-experimental strategy is implemented to design the microfluidic device and optimize the capturing efficiency and to maximize the throughput. The proposed design enables high CE and purity of target cells and real-time time on-chip monitoring of their behavior. The strategy introduced in this paper offers a simple and low-cost yet robust opportunity for early-stage diagnosis and monitoring of cancer-associated biomarkers.

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.

A microfluidic immunosensor for automatic detection of carcinoembryonic antigen based on immunomagnetic separation and droplet arrays.

Diagnosis of cancer by biomarkers plays an important role in human health and life. However, current laboratory techniques for detecting cancer biomarkers still require laborious and time-consuming operation by skilled operators and associated laboratory instruments. This work presents a colorimetric biosensor for the rapid and sensitive detection of carcinoembryonic antigen (CEA) based on an automated immunomagnetic separation platform and a droplet array microfluidic chip with the aid of an image analysis system. Immunomagnetic nanoparticles (MNPs) were used to capture CEA in the samples. CEA-detecting antibodies and horseradish peroxidase (HRP) were modified on polystyrene microspheres (PS), catalysing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) as signal outputs. Color reaction data were analyzed to establish a CEA concentration standard curve. The movement of MNPs between droplets in the microfluidic chip is achieved using an automatically programmable magnetic control system. This colorimetric biosensor has been used for the simultaneous detection of six CEA samples ranging from 100 pg mL-1 to 100 ng mL-1 with a detection limit of 14.347 pg mL-1 in 10 min, following the linear equation: y = -4.773 ln(x) + 156.26 with a correlation of R2 = 0.9924, and the entire workflow can be completed within 80 minutes. The microfluidic immunosensor designed in this paper has the advantages of low cost, automation, low sample consumption, high throughput, and promising applications in biochemistry.

Immunomagnetic separation coupled with flow cytometry for the analysis of Legionella pneumophila in aerosols.

Legionella pneumophila are pathogenic bacteria that can be found in high concentrations in artificial water systems like evaporative cooling towers, which have been the source of frequent outbreaks in recent years. Since inhaled L. pneumophila can lead to Legionnaires' disease, the development of suitable sampling and rapid analysis strategies for these bacteria in aerosols is therefore of great relevance. In this work, different concentrations of viable L. pneumophila Sg 1 were nebulized and sampled by the cyclone sampler Coriolis® µ under defined conditions in a bioaerosol chamber. To quantify intact Legionella cells, the collected bioaerosols were subsequently analyzed by immunomagnetic separation coupled with flow cytometry (IMS-FCM) on the platform rqmicro.COUNT. For analytical comparison, measurements with qPCR and cultivation were performed. Limits of detection (LOD) of 2.9 × 103 intact cells m-3 for IMS-FCM and 7.8 × 102 intact cells m-3 for qPCR indicating a comparable sensitivity as in culture (LOD = 1.5 × 103 culturable cells m-3). Over a working range of 103 - 106 cells mL-1, the analysis of nebulized and collected aerosol samples with IMS-FCM and qPCR provides higher recovery rates and more consistent results than by cultivation. Overall, IMS-FCM is a suitable culture-independent method for quantification of L. pneumophila in bioaerosols and is promising for field application due to its simplicity in sample preparation.

Isolation of Fusobacterium nucleatum from human feces using immunomagnetic separation coupled with fastidious anaerobe agar.

AIM: Fusobacterium nucleatum (F. nucleatum) is associated with the initiation, development, and metastasis of colorectal cancer. However, it is difficult to isolate F. nucleatum from clinical specimens. In this study, we aimed to develop an effective and rapid method for isolating F. nucleatum from human feces using polyclonal antibody (PAB)-coated immunomagnetic beads (IMBs) with selective media. METHODS AND RESULTS: IMBs conjugated with PAB were prepared and used to isolate F. nucleatum from human feces, and the bacteria were cultured with selective culture media (fastidious anaerobe agar + nalidixic acid + vancomycin). Under optimized experimental conditions, IMBs could selectively recover F. nucleatum from fecal microbiota samples spiked with Peptostreptococcus or Bacteroides fragilis. In artificial fecal samples, the detection sensitivity of IMBs for F. nucleatum was 103 CFU mL-1. In addition, IMBs combined with selective media could rapidly isolate F. nucleatum from human feces. CONCLUSIONS: This study successfully established an effective method for the rapid isolation of F. nucleatum from human feces by IMBs. The whole procedure requires 2-3 days, and has a sensitivity of 103 CFU mL-1 feces.

Comparison of four commercial immunomagnetic separation kits for the detection of Cryptosporidium.

Cryptosporidium spp. are protozoan parasites of significant health importance found in environmental waters globally. Four commercially available Cryptosporidium-specific immunomagnetic separation (IMS) kits used in various water sample matrices were analysed and compared. Beads were characterised by flow cytometry and tested for the recovery efficiencies for oocysts spiked into different matrices: river water sediment, clay sample, and filter backwash sample. Results showed that Dynabeads™ Cryptosporidium and Waterborne Crypto-Grab™ kits contained immunoglobulin IgM antibody-coated beads. In contrast, the BioPoint CryptoBead and the TCS Isolate kits contained immunoglobulin IgG antibody-coated beads. BioPoint CryptoBead was significantly coated with more antibodies and were able to capture oocysts more rapidly compared to the other beads. Recovery efficiencies of Dynabeads™, TCS Isolate® beads, and BioPoint CryptoBead ranged from 55 to 93% when tested against different sample matrices, with BioPoint CryptoBead resulting in the highest at 93% in reagent-grade water and Dynabeads™ at 55%, the lowest against clay samples. The Waterborne beads did not perform well on any samples, with recovery efficiencies ranging from 0 to 8%. Fluorescence microscopy analyses showed that both the IMS method and the sample matrix processed affect the quality of the membranes, with the cleanest samples for microscopy examination observed from BioPoint CryptoBead.

Targeting Novel LPXTG Surface Proteins with Monoclonal Antibodies for Immunomagnetic Separation of Listeria monocytogenes.

The Gram-positive bacterium Listeria monocytogenes causes a significantly high percentage of fatalities among human foodborne illnesses. Surface proteins, specifically expressed from a wide range of L. monocytogenes serotypes under selective enrichment culture conditions, can serve as targets for the isolation of this pathogen using antibody-based methods to facilitate molecular detection. In this study, monoclonal antibodies (MAbs), previously raised against the L. monocytogenes LPXTG surface proteins LMOf2365_0639 and LMOf2365_0148, were investigated for their ability to isolate L. monocytogenes from bacterial samples with immunomagnetic separation (IMS). Only 1 out of 35 MAbs against LMOf2365_0639, M3644, was capable of capturing L. monocytogenes. Among all the 24 MAbs examined against LMOf2365_0148, 4 MAbs, M3686, M3697, M3699, and M3700, were capable of capturing L. monocytogenes cells specifically from abbreviated primary selective enrichment cultures in either Palcam or LEB/UVM1 media or from mixed samples containing target and nontarget bacteria. MAb M3686 showed a unique specificity with the capability to capture strains of seven L. monocytogenes serotypes (1/2a, 1/2b, 1/2c, 3a, 4a, 4b, and 4d). These promising MAbs were subsequently characterized by quantitative measurements of antigen-binding affinity using surface plasmon resonance analysis and epitope mapping using overlapping recombinant polypeptides. The usefulness of these MAbs to LMOf2365_0148 in bacterial capture was consistent with their high affinities with KD constants in the nanomolar range and can be explored further for the development of an automated IMS method suitable for routine isolation of L. monocytogenes from food and environmental samples.

Separation and colorimetric detection of Escherichia coli by phage tail fiber protein combined with nano-magnetic beads.

A colorimetric detection method for Escherichia coli (E. coli) in water was established based on a T7 phage tail fiber protein-magnetic separation. Firstly, the tail fiber protein (TFP) was expressed and purified to specifically recognize E. coli, which was verified by using fusion protein GFP-tagged TFP (GFP-TFP) and fluorescence microscopy. Then TFP conjugated with magnetic beads were applied to capture and separate E. coli. The TFP was covalently immobilized on the surface of magnetic beads and captured E. coli as verified by scanning electron microscopy (SEM). Finally, polymyxin B was used to lyse E. coli in solution and the released intracellular ß-galactosidase (ß-gal) could hydrolyze the colorimetric substrate chlorophenol red-ß-D-galactopyranoside (CPRG), causing color change from yellow to purple. The high capture efficiencies of E. coli ranged from 88.70% to 95.65% and E. coli could be detected at a concentration of 102 CFU/mL by naked eyes. The specificity of the chromogenic substrate was evaluated using five different pathogen strains as competitors and tests with four kinds of real water samples showed recoveries of 86.00% to 92.25%. The colorimetric changes determined by visual inspection can be developed as an efficient platform for point-of-care detection of E. coli in resource-limited regions.

Fully Integrated Microfluidic Biosensor with Finger Actuation for the Ultrasensitive Detection of Escherichia coli O157:H7.

A portable microfluidic biosensor was developed for the detection of E. coli O157:H7 using finger actuation. The chip was assembled with three functional zones, immunomagnetic separation, nucleic acid extraction and purification, and signal detection. First, antibody-modified magnetic nanoparticles (MNPs) were used to separate the target bacteria from the sample. The captured bacteria were then lysed and silica-coated MNPs were used to absorb DNA, followed by washing and eluting to obtain purified DNA. The obtained DNA was subjected to amplification and fluorescence detection based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeat-associated protein/Cas12a reaction. The fluorescence images were collected and analyzed using a smartphone app under a 3D-printed detection device. It could quantitatively detect E. coli O157:H7 from 102 to 108 CFU/mL in 2.5 h with a limit of detection (LOD) of 10 CFU/mL. The recovery rate ranged from 104 to 120%. Overall, the biosensor realizes "sample-in and answer-out" assay for E. coli O157:H7 and eliminates the need for external pumps and skilled personnel.

Controllable Environment Protein Corona-Disguised Immunomagnetic Beads for High-Performance Circulating Tumor Cell Enrichment.

The enrichment performance of immunomagnetic beads (IMBs) in blood samples is usually challenging due to the ungoverned, in situ-formed protein corona, as it generally leads to negative effects, such as impeded targeting capacity and unwanted nonspecific absorption. On the contrary, a controlled protein premodification of IMBs with diverse functional environment (blood) proteins endows the composites with a new biological identity and may improve the anti-nonspecific ability, resulting in promising isolation benefits for circulating tumor cell (CTC) enrichment and downstream analyses. Specifically, fetal bovine serum and the four most abundant blood proteins, including human serum albumin, fibrinogen, immunoglobulin, and transferrin, were separately applied in this work. Conclusively, the biological properties of the applied protein corona camouflage have a great influence on the capture performance of IMBs, and certain proteins can enhance the enrichment performance to a large extent. Promisingly, human serum albumin-camouflaged IMBs (HSA-PIMBs) achieved a capture efficiency of 84.0-90.0% and significantly minimized nonspecific absorbed leukocytes to 164-264 in blood samples (0.5 mL, 25-55 model CTCs). Furthermore, HSA-PIMBs isolated 62-505 CTCs and 13-31 leukocytes from the blood samples of five cancer patients. The novel environment camouflage strategy provides a new insight into protein corona utilization and may improve the performance of targeted nanomaterials in a complex biological environment.

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.

Outer membrane protein A (OmpA) may be used as a novel target to enrich and detect Escherichia coli in milk samples.

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 100 cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.

Multigene model for predicting metastatic prostate cancer using circulating tumor cells by microfluidic magnetophoresis.

We aimed to isolate circulating tumor cells (CTCs) using a microfluidic technique with a novel lateral magnetophoretic microseparator. Prostate cancer-specific gene expressions were evaluated using mRNA from the isolated CTCs. A CTC-based multigene model was then developed for identifying advanced prostate cancer. Peripheral blood samples were obtained from five healthy donors and patients with localized prostate cancer (26 cases), metastatic hormone-sensitive prostate cancer (mHSPC, 10 cases), and metastatic castration-resistant prostate cancer (mCRPC, 28 cases). CTC recovery rate and purity (enriched CTCs/total cells) were evaluated according to cancer stage. The areas under the curves of the six gene expressions were used to evaluate whether multigene models could identify mHSPC or mCRPC. The number of CTCs and their purity increased at more advanced cancer stages. In mHSPC/mCRPC cases, the specimens had an average of 27.5 CTCs/mL blood, which was 4.2 × higher than the isolation rate for localized disease. The CTC purity increased from 2.1% for localized disease to 3.8% for mHSPC and 6.7% for mCRPC, with increased CTC expression of the genes encoding prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and cytokeratin 19 (KRT19). All disease stages exhibited expression of the genes encoding androgen receptor (AR) and epithelial cell adhesion molecule (EpCAM), although expression of the AR-V7 variant was relatively rare. Relative to each gene alone, the multigene model had better accuracy for predicting advanced prostate cancer. Our lateral magnetophoretic microseparator can be used for identifying prostate cancer biomarkers. In addition, CTC-based genetic signatures may guide the early diagnosis of advanced prostate cancer.

Development of an optimal protocol for molecular profiling of tumor cells in pleural effusions at single-cell level.

Liquid biopsy analyzes the current status of primary tumors and their metastatic regions. We aimed to develop an optimized protocol for single-cell sequencing of floating tumor cells (FTCs) in pleural effusion as a laboratory test. FTCs were enriched using a negative selection of white blood cells by a magnetic-activated cell sorting system, and CD45-negative and cytokeratin-positive selection using a microfluidic cell separation system with a dielectrophoretic array. The enriched tumor cells were subjected to whole-genome amplification (WGA) followed by genome sequencing. The FTC analysis detected an EGFR exon 19 deletion in Case 1 (12/19 cells, 63.2%), and EML4-ALK fusion (17/20 cells, 85%) with an alectinib-resistant mutation of ALK (p.G1202R) in Case 2. To eliminate WGA-associated errors and increase the uniformity of the WGA product, the protocol was revised to sequence multiple single FTCs individually. An analytical pipeline, accurate single-cell mutation detector (ASMD), was developed to identify somatic mutations of FTCs. The large numbers of WGA-associated errors were cleaned up, and the somatic mutations detected in FTCs by ASMD were concordant with those found in tissue specimens. This protocol is applicable to circulating tumor cells analysis of peripheral blood and expands the possibility of utilizing molecular profiling of cancers.