Current Methods for Extraction and Concentration of Foodborne Bacteria with Glycan-Coated Magnetic Nanoparticles: A Review.

Rapid and accurate food pathogen detection is an essential step to preventing foodborne illnesses. Before detection, removal of bacteria from the food matrix and concentration to detectable levels are often essential steps. Although many reviews discuss rapid concentration methods for foodborne pathogens, the use of glycan-coated magnetic nanoparticles (MNPs) is often omitted. This review seeks to analyze the potential of this technique as a rapid and cost-effective solution for concentration of bacteria directly from foods. The primary focus is the mechanism of glycan-coated MNP binding, as well as its current applications in concentration of foodborne pathogens. First, a background on the synthesis, properties, and applications of MNPs is provided. Second, synthesis of glycan-coated particles and their theorized mechanism for bacterial adhesion is described. Existing research into extraction of bacteria directly from food matrices is also analyzed. Finally, glycan-coated MNPs are compared to the magnetic separation technique of immunomagnetic separation (IMS) in terms of cost, time, and other factors. At its current state, glycan-coated MNPs require more research to fully identify the mechanism, potential for optimization, and extraction capabilities directly in food matrices. However, current research indicates glycan-coated MNPs are an incredibly cost-effective method for rapid food pathogen extraction and concentration.

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.

Magnetophoretic Micro-Distributor for Controlled Clustering of Cells.

Cell clustering techniques are important to produce artificial cell clusters for in vitro models of intercellular mechanisms at the single-cell level. The analyses considering physical variables such as the shape and size of cells have been very limited. In addition, the precise manipulation of cells and control of the physical variables are still challenging. In this paper, a magnetophoretic device consisting of a trampoline micromagnet and active elements that enable the control of individual selective jumping motion and positioning of a micro-object is proposed. Based on a numerical simulation under various conditions, automatic separation or selective clustering of micro-objects according to their sizes is performed by parallel control and programmable manipulation. This method provides efficient control of the physical variables of cells and grouping of cells with the desired size and number, which can be a milestone for a better understanding of the intercellular dynamics between clustered cells at the single-cell level for future cell-on-chip applications.

Visual and quantitative detection of E. coli O157:H7 by coupling immunomagnetic separation and quantum dot-based paper strip.

Simple and visual quantitative detection of foodborne pathogens can effectively reduce the outbreaks of foodborne diseases. Herein, we developed a simple and sensitive quantum dot (QD)-based paper device for visual and quantitative detection of Escherichia coli (E. coli) O157:H7 based on immunomagnetic separation and nanoparticle dissolution-triggered signal amplification. In this study, E. coli O157:H7 was magnetically separated and labeled with silver nanoparticles (AgNPs), and the AgNP labels can be converted into millions of Ag ions, which subsequently quench the fluorescence of QDs in the paper strip, which along with the readout can be visualized and quantified by the change in length of fluorescent quenched band. Owing to the high capture efficiency and effective signal amplification, as low as 500 cfu mL-1 of E. coli O157:H7 could be easily detected by naked eyes. Furthermore, this novel platform was successfully applied to detect E. coli O157:H7 in spiked milk samples with good accuracy, indicating its potential in the detection of foodborne pathogens in real samples.

Norovirus Extraction from Frozen Raspberries Using Magnetic Silica Beads.

Human noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.

Detection of toxin B of Clostridium difficile based on immunomagnetic separation and aptamer-mediated colorimetric assay.

Clostridium difficile can cause antibiotic-associated diarrhoea or pseudo-membranous colitis in humans and animals. Currently, the various methods such as microbiological culture, cytotoxic assay, ELISA and polymerase chain reaction have been used to detect Clostridium difficile infection (CDI). These conventional methods, however, require long detection time and professional staff. The paper is to describe a simple strategy which employs immunomagnetic separation and aptamer-mediated colorimetric assay for the detection of toxin B of C. difficile (TcdB) in the stool samples. HRP-labelled aptamer against TcdB selected by SELEX was firstly captured on the surface of magnetic beads (MB) by DNA hybridization with a complementary strand. In the presence of TcdB, aptamer specifically recognized and bound TcdB, disturbing the DNA hybridization and causing the release of HRP-aptamer from MB. This reduced the catalytic capacity of HRP and consequently the absorption intensity. As there was a relationship between the decrease in the absorption intensity and target concentration, a quantitative analysis of TcdB can be accomplished by the measurement of the absorption intensity. Under the optimal conditions, the assay system is able to detect TcdB at a concentration down to 5 ng ml-1 . Moreover the method had specificity of 97% and sensitivity of 66% and the system remained excellent stability within 4 weeks. The proposed method is a valuable screening procedure for CDI and can be extended readily to detection of other clinically important pathogens.

Colorimetric Detection of Escherichia coli O157:H7 with Signal Enhancement Using Size-Based Filtration on a Finger-Powered Microfluidic Device.

Although immunomagnetic separation is a useful sample pretreatment method that can be used to separate target pathogens from a raw sample, it is challenging to remove unbound free magnetic nanoparticles (MNPs) for colorimetric detection of target pathogens. Here, size-based filtration was exploited for the rapid on-site detection of pathogens separated by immunomagnetic separation in order to remove unbound free MNPs using a finger-powered microfluidic device. A membrane filter and an absorbent pad were integrated into the device and a mixture of unbound free MNPs and MNP-bound Escherichia coli (E. coli) O157:H7 was dispensed over the membrane filter by pressing and releasing the pressure chamber. A colorimetric signal was generated by MNP-bound E. coli O157:H7 while unbound free MNPs were washed out by the absorbent. Furthermore, the colorimetric signals can be amplified using a gold enhancer solution when gold-coated MNPs were used instead of MNPs. As a result, 102 CFU/mL E. coli O157:H7 could be detected by the enhanced colorimetric signal on a proposed device.

Isolation of Trypanosoma brucei brucei Infection-Derived Splenic Marginal Zone B Cells Based on CD1dHigh/B220High Surface Expression in a Two-Step MACS-FACS Approach.

Magnetic- and fluorescent-activated cell sorting (MACS and FACS) are used for isolation of distinct cell populations for subsequent studies including transcriptomics. The latter allows for the analysis of infection-induced alterations in gene expression profiles. MACS and FACS both use antibodies against cell surface molecules to isolate populations of interest. Standardized methods for both approaches exist for use in mouse models. These protocols, however, do not account for the fact that infection-associated immunopathology can significantly modulate the cell surface expression of targeted molecules. This is the case for Trypanosoma brucei brucei infection, where downregulation of CD23 surface expression on B cells has been reported. This hallmark of progressing infection interferes with the commercially available MACS technique for B cell purification, as CD23 expression is the target for the separation between Marginal Zone (MZ) and Follicular (Fo) B cells. Here, we provide a robust alternative method for isolation of infection-derived MZ B cells using CD1d and B220 surface molecules in a two-step MACS-FACS approach. The method yields 99% pure viable infection-derived MZ B cells, allowing extraction of a high quality total RNA suitable for subsequent RNA sequencing.

Detection of Salmonella Enterica in Egg Yolk by PCR on a Microfluidic Disc Device Using Immunomagnetic Beads.

Salmonella enterica is a pathogenic bacterium that causes foodborne illness. One of the vehicle foods of S. enterica are chicken eggs. Efficient collection of the bacterium is necessary to detect it specifically. We developed a method to detect S. enterica by PCR on a microfluidic disc device using a fluorescent probe. Salmonella enterica cells were isolated in the microchambers on the device, followed by thermal lysis and PCR targeting with the invA gene, a gene specific to S. enterica, were observed by measurement of the fluorescent signal that resulted from gene amplification. However, the developed method was unable to discriminate viable cells from dead cells. Consequently, in this study, magnetic beads modified with anti-Salmonella antibody were utilized to detect viable Salmonella cells from egg yolk prior to PCR on the device. While using the antibody-modified beads, egg yolk components, which inhibit PCR, were removed. The collected cells were subsequently detected by PCR of the invA gene on a microfluidic disc device. This method enabled the detection of viable cells without the inhibition of PCR by any egg component. S. enterica was detected at 5.0×104 cells mL-1 or at a higher concentration of egg yolk within 6 h including the sampling time.

Development of polyclonal-antibody-coated immunomagnetic beads for separation and detection of koi herpesvirus in large-volume samples.

To separate and concentrate koi herpesvirus (KHV) from large-volume samples, a separation method based on immunomagnetic beads (IMBs) coated with polyclonal antibody directed against KHV was developed. After treatment with IMBs, viral DNA was extracted from samples and used as a template for quantitative PCR (qPCR). The results showed that the concentration of the template DNA extracted from the virus that had been separated using IMBs was 9.65-fold higher than that from virus not treated with IMBs. The detection limit of the IMBs/qPCR method was found to be at least 10 times lower than that of qPCR alone.

Quality control of multiplex antibody detection in samples from large-scale surveys: the example of malaria in Haiti.

Measuring antimalarial antibodies can estimate transmission in a population. To compare outputs, standardized laboratory testing is required. Here we describe the in-country establishment and quality control (QC) of a multiplex bead assay (MBA) for three sero-surveys in Haiti. Total IgG data against 21 antigens were collected for 32,758 participants. Titration curves of hyperimmune sera were included on assay plates, assay signals underwent 5-parameter regression, and inspection of the median and interquartile range (IQR) for the y-inflection point was used to determine assay precision. The medians and IQRs were similar for Surveys 1 and 2 for most antigens, while the IQRs increased for some antigens in Survey 3. Levey-Jennings charts for selected antigens provided a pass/fail criterion for each assay plate and, of 387 assay plates, 13 (3.4%) were repeated. Individual samples failed if IgG binding to the generic glutathione-S-transferase protein was observed, with 659 (2.0%) samples failing. An additional 455 (1.4%) observations failed due to low bead numbers (<20/analyte). The final dataset included 609,438 anti-malaria IgG data points from 32,099 participants; 96.6% of all potential data points if no QC failures had occurred. The MBA can be deployed with high-throughput data collection and low inter-plate variability while ensuring data quality.

Portable Resource-Independent Blood-Plasma Separator.

The centrifuge is the gold standard for lab-based sample processing. While extremely efficient and robust, centrifuges are seldom used in the field due to the high-power requirements, size, and operational complexity. The lack of viable alternatives for remote sample collection has crippled the ability for mobile practitioners in human and animal medicine to reliably collect blood samples from their patients. There is no truly resource-independent solution that is able to perform highly efficient blood-plasma separation. Here, we describe our initial efforts in developing the High Efficiency Rapid Magnetic Erythrocyte Separator (H.E.R.M.E.S) sleeve, an apparatus that uses a magnetic bead-based separation assay in a scaled-up form factor to achieve highly efficient separation of erythrocytes from plasma within a short amount of time. The sleeve is easy-to-use, is completely resource independent, and achieves highly efficient separation in sample volumes as large as 1 mL by means of a unique mixing scheme. We demonstrate the performance of the sleeve with human blood samples and compare it against conventional end-over-end mixing.

Lysin cell-binding domain-functionalized magnetic beads for detection of Staphylococcus aureus via inhibition of fluorescence of Amplex Red/hydrogen peroxide assay by intracellular catalase.

Accurate and rapid identification of Staphylococcus aureus (S. aureus) is of great significance for controlling the food poisoning and infectious diseases caused by S. aureus. In this study, a novel strategy that combines lysin cell-binding domain (CBD)-based magnetic separation with fluorescence detection was developed for the specific and sensitive quantification of S. aureus in authentic samples. The S. aureus cells were separated from the sample matrix by lysin CBD-functionalized magnetic beads. Following lysis by lysostaphin, intracellular catalase was released from S. aureus cells and detected by a fluorometric system composed of horseradish peroxidase (HRP), hydrogen peroxide (H2O2), and Amplex Red. S. aureus was quantified via the inhibitory effect of the released intracellular catalase on the fluorometric system since the catalase could decompose the H2O2. Optimized conditions afforded a calibration curve for S. aureus ranging from 1.0 × 102 to 1.0 × 107 CFU mL-1. The detection limit was as low as 78 CFU mL-1 in phosphate-buffered saline (PBS), and the total detection process could be completed in less than 50 min. Other bacteria associated with common food-borne and nosocomial infections negligibly interfered with S. aureus detection, except for Staphylococcus epidermidis, which may have slightly interfered. Moreover, the potential of this proposed method for practical applications has been demonstrated by detection assays of sterilized milk and human serum. Graphical abstract.

In Vitro Detection of Cellular Adjuvant Properties of Human Invariant Natural Killer T Cells.

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that play a crucial role in the tumor surveillance. The activation of iNKT cells by their specific ligand α-galactosylceramide (α-GalCer) induces the activation of dendritic cells (DCs) via reciprocal interaction, which results in the generation of cellular immunity against cancer. Here we describe a method to detect DC-mediated cellular adjuvant properties of human iNKT cells in vitro.

Clonogenic Culture of Mouse Thymic Epithelial Cells.

The thymus plays an essential role in the development and selection of T cells by providing a unique microenvironment that is mainly composed of thymic epithelial cells (TECs). We previously identified stem cells of medullary TECs (mTECs) that are crucial for central tolerance induction using a novel clonogenic culture system. We also found that medullary thymic epithelial stem cells (mTESCs) maintain life-long mTECs regeneration and central T cell self-tolerance in mouse models. The clonogenic efficiency of TECs in vitro is highly correlated to the TEC reconstitution activity in vivo. Here, we describe the clonogenic culture system to evaluate the self-renewing activity of TESCs. The colonies are derived from TESCs, are visualized and quantified by rhodamine-B staining on a feeder layer, and can be passaged in vitro. Thus, our system enables quantitative evaluation of TESC activity and is useful for dissecting the mechanisms that regulate TESC activity in physiological aging as well as in various clinical settings.

Electronic profiling of membrane antigen expression via immunomagnetic cell manipulation.

Membrane antigens control cell function by regulating biochemical interactions and hence are routinely used as diagnostic and prognostic targets in biomedicine. Fluorescent labeling and subsequent optical interrogation of cell membrane antigens, while highly effective, limit expression profiling to centralized facilities that can afford and operate complex instrumentation. Here, we introduce a cytometry technique that computes surface expression of immunomagnetically labeled cells by electrically tracking their trajectory under a magnetic field gradient on a microfluidic chip with a throughput of >500 cells per min. In addition to enabling the creation of a frugal cytometry platform, this immunomagnetic cell manipulation-based measurement approach allows direct expression profiling of target subpopulations from non-purified samples. We applied our technology to measure epithelial cell adhesion molecule expression on human breast cancer cells. Once calibrated, surface expression and size measurements match remarkably well with fluorescence-based measurements from a commercial flow cytometer. Quantitative measurements of biochemical and biophysical cell characteristics with a disposable cytometer have the potential to impact point of care testing of clinical samples particularly in resource limited settings.

Rapid prototyping of Nanoroughened polydimethylsiloxane surfaces for the enhancement of immunomagnetic isolation and recovery of rare tumor cells.

Traditional immunomagnetic assays for the isolation and recovery of circulating tumor cells (CTCs) usually require sophisticated device or intense magnetic field to simultaneously achieve high capture efficiency and high throughout. In this study, a simple microfluidic chip featured with nanoroughened channel substrate was developed for effectively capture and release of CTCs based on an immunomagnetic chip-based approach. The nanoroughened substrate aims to increase the cell-surface contact area, facilitate the immobilization of magnet particles (MPs) and accommodate cell attachment tendency. Hep3B tumor cells were firstly conjugated with MPs that were functionalized with anti-EpCAM. Comparing with the flat channel, MPs modified tumor cells can be more effectively captured on nanoroughened substrate at the presence of the magnetic field. Upon the removal of magnetic field, these captured cells can be released from the device and collected for further analysis. Under the optimum operating conditions, the capture efficiency of tumor cells was obtained as high as ~90% with a detection limit of 10 cell per mL. Additionally, recovery rates of trapped tumor cells at various densities all exceeded 90% and their biological potencies were well retained by investigating the cell attachment and proliferation. Therefore, the present approach may potentially be used in clinical CTC analysis for cancer diagnosis and prognosis as well as the fundamental understanding of tumor metastasis.

Magnetic nanocomposites: versatile tool for the combination of immunomagnetic separation with flow-based chemiluminescence immunochip for rapid biosensing of Staphylococcal enterotoxin B in milk.

Immunomagnetic separation (IMS) was combined with flow-based chemiluminescence sandwich immunoassays (CL-SIA) for the quantification of Staphylococcal enterotoxin B in milk. Therefore, iron oxide-shell silica-core magnetic nanocomposites were conjugated to biotinylated anti-SEB antibodies (MNC-IgGs). MNC-IgGs were applied successfully for (i) capturing SEB in milk samples by an affinity reaction, (ii) magnetophoretic collection on antibody spots in a channel of a flow-based immunochip, and (iii) sensitive enzymatic chemiluminescence detection of biotin labels by poly(horseradish peroxidase)-streptavidin. IMS was performed in 0.6 mL and 100 mL milk samples resulting in detection limits of 50 ng L-1 and 0.39 ng L-1, respectively, for the combined analytical method. It was shown that the assay sensitivity was dramatically improved by the combination of IMS with flow-based CL-SIA compared to CL-SIA directly applied with milk samples (detection limit 130 ng L-1). The IMS-CL-SIA has a time-to-result of 2-3 h. The reported combined analytical method can be used for a rapid control of SEB in complex food matrices such as milk. In future, even the monitoring of multiple contaminants in food or water may be performed by IMS-CL-SIA. Graphical abstract.

An integrated magnetic microfluidic chip for rapid immunodetection of the prostate specific antigen using immunomagnetic beads.

The authors describe an integrated microfluidic chip for immunodetection of the prostate specific antigen (PSA) by using giant magnetoimpedance (GMI) sensor. This chip contains an immunoreaction platform and a biomarker detection system. The immunoreaction platform contains an incubation chamber and a reactive chamber to implement immunological reaction in microfluidics. The system can detect PSA rapidly with ultra-high sensitivity. Both are fabricated by MEMS technology. Immunomagnetic beads (If PSA binds to its antibody (that is labeled with immunomagnetic beads; IMBs) it will be trapped on the surface of self-assembled film. Trapped IMBs generate a stray magnetic field under the magnetization of the external applied magnetic field and can be detected by the GMI sensor. The chip can detect PSA with a detection limit as low as 0.1 ng ∙ mL-1 and works in the 0.1 ng ∙ mL-1 to 20 ng ∙ mL-1 concentration range. Compared to established GMI biosensors, the magnetic microfluidic chip reduces assay time, and lends itself to fast detection. It also avoids complex handling steps, enhances reaction efficiency and decreases experimental errors. Graphical abstract An integrated magnetic microfluidic chip which contains immunoreaction platform and biomarker detection system was designed and microfabricated by micro-electromechanical systems (MEMS) technology to detect prostate specific antigen (PSA) rapidly, and has promise in Point-of-care (PoC) diagnostic applications.

A flyover style microfluidic chip for highly purified magnetic cell separation.

White blood cells (WBCs) isolated from peripheral blood have been verified as important biomarkers for the diagnosis, treatment and prognosis of cancer. However, it's still under challenge to acquire high-purity WBCs, even by taking advantage of current microfluidic technology. Considering the universality of clinical magnetic activated cell sorting (MACS) method, new developments on microfluidic chip in combination of magnetic cells separation technologies may provide a fascinating approach for high-purity WBCs sorting and widely clinical application. Here, we present a flyover style microfluidic chip which has been elaborately embedded with two-stage magnetic separation in continuous flow for WBCs sorting. Immunomagnetic micro/nano-particles (IMNPs) labeled WBC (WBC@IMNPs) were sequentially separated by a lateral magnetic force and a vertical magnetic force, and the final separation purity of WBCs reached up to 93 ±â€¯1.67% at a flow rate of 20 µL min-1. Furthermore, the WBCs viability was up to 97.5 ±â€¯1.8%. Consequently, this novel flyover style microfluidic-chip with magnetic separation technology has been successfully demonstrated as cut-in-edge method for high-purity WBCs sorting, and obviously it's easy to extend for other types of cells sorting under great potential application in biomedical fields.