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.

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.

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.

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.

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.

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.

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.

Improving the Sensitivity of Fluorescence-Based Immunoassays by Photobleaching the Autofluorescence of Magnetic Beads.

In fluorescence-based assays, usually a target molecule is captured using a probe conjugated to a capture surface, and then detected using a second fluorescently labeled probe. One of the most common capture surfaces is a magnetic bead. However, magnetic beads exhibit strong autofluorescence, which often overlaps with the emission of the reporter fluorescent dyes and limits the analytical performance of the assay. Here, several widely used magnetic beads are photobleached and their autofluorescence is reduced to 1% of the initial value. Their autofluorescence properties, including their photobleaching decay rates and autofluorescence spectra pre- and post-photobleaching, and the stability of the photobleaching over a period of two months are analyzed. The photobleached beads are stable over time and their surface functionality is retained. In a high-sensitivity LX-200 system using photobleached magnetic beads, human interleukin-8 is detected with a threefold improvement in detection limit and signal-to-noise ratio over results achievable with nonbleached beads. Since many contemporary immunoassays rely on magnetic beads as capture surfaces, prebleaching the beads may significantly improve the analytical performance of these assays. Moreover, nonmagnetic beads with low autofluorescence are also successfully photobleached, suggesting that photobleaching can be applied to various capture surfaces used in fluorescence-based assays.

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.

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.

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.

Advanced immunocapture of milk-borne Salmonella by microfluidic magnetically stabilized fluidized bed.

The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (µMSFB), developed for the capture and on-chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti-Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti-Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the µMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.

Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells.

BACKGROUND AIMS: For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products. METHODS: Unstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity. RESULTS: Selections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained. DISCUSSION: The novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity.

Combined immunomagnetic capture coupled with ultrasensitive plasmonic detection of circulating tumor cells in blood.

We demonstrate enhanced on-chip circulating tumor cell (CTC) detection through the incorporation of plasmonic-enhanced near-infrared (NIR) fluorescence screening. Specifically, the performance of plasmonic gold coated chips was evaluated on our previously reported immunomagnetic CTC capture system and compared to the performance of a regular chip. Three main performance metrics were evaluated: capture efficiency, capture reproducibility, and clinical efficacy. Use of the plasmonic chip to capture SK-BR-3 cells in PBS, resulted in a capture efficiency of 82%, compared to 76% with a regular chip. Both chips showed excellent capture reproducibility for all three cells lines evaluated (MCF-7, SK-BR-3, Colo 205) in both PBS and peripheral blood, with R2 values ranging from 0.983 to 0.996. Finally, performance of the plasmonic chip was evaluated on thirteen peripheral blood samples in patients with both breast and prostate cancer. The regular chip detected 2-8 cells per 5 mL of blood, while the plasmonic chip detected 8-85 cells per 5 mL of blood in parallel samples. In summary, we successfully demonstrate improved CTC capture and detection capabilities through use of plasmonic-enhanced near-infrared (NIR) fluorescence screening in both in vitro and ex vivo experiments. This work not only has the potential to improve clinical outcomes though improved CTC analysis, but also demonstrates successful interface design between plasmonic materials and cell capture for bioanalytical applications.

Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus.

Single-domain antibody (sdAb) or nanobody possesses specific features non-accessible for conventional antibodies that make them suitable for research and biotechnological applications. Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets, resulting in great economic losses all over the world. To detect and isolate PEDV rapidly and accurately is important for the control and further research of the clinical PEDV strains. In this study, four sdAb fragments (sdAb-Mc19/29/30/37) targeting the membrane (M) protein of PEDV were selected from sdAb library that was constructed through M protein-immunized Camelus bactrianus. The selected sdAb-Mcs were solubly expressed in Escherichia coli. The functional characteristics analysis revealed that the recombinant sdAb-Mcs have excellent binding activity and specificity to M protein but have no neutralizing activity to PEDV. For further application, sdAb-Mc37 was conjugated with quantum dots to synthesize a nanoprobe for imaging PEDV in vero cells. The observed fluorescence in vero cells clearly reflects that PEDV virions can be reliably recognized and labeled by the nanoprobe. Furthermore, the sdAb-Mc29 was conjugated with superparamagnetic nanobeads to construct immunomagnetic nanobeads (IMNBs) used to isolate PEDV. One PEDV strain was successfully isolated from clinical fecal sample, suggesting IMNBs as a novel and efficient tool suitable for PEDV isolation from clinical samples. This study provided a novel application and substantiated the suitability of sdAb as a specific binder for the isolation of viruses.

Purification of Fluorescent Labeled Cells from Dissociated Ciona Embryos.

Genome-wide studies in Ciona often require highly purified cell populations. In this methods chapter, we introduce multi-channel combinatorial fluorescence activated cells sorting (FACS) and magnetic-activated cell sorting (MACS) as two sensitive and efficient tools for isolating lineage-specific cell populations from dissociated Ciona embryos and larvae. We present isolation of trunk ventral cell (TVC) progeny as the test case most commonly used in our laboratory. These approaches may also be applied to purify other cell populations with the proper combination of tissue-specific reporters.

Detection of Cronobacter species in powdered infant formula using immunoliposome-based immunomagnetic concentration and separation assay.

Cronobacter species are foodborne pathogens that can affect the human central nervous system. Survivors of Cronobacter infections often suffer from severe neurological impairments, including hydrocephalus, quadriplegia, and developmental delays in all ages, especially in infants and the immunocompromised. Moreover, Cronobacter species pose a high risk in powdered infant formula (PIF) because PIF is a major source of nutrition for infants worldwide. To develop a rapid and sensitive detection method for Cronobacter species in PIF, immunoliposomes and immunomagnetic nanoparticles were synthesized, after which an immunoliposome-based immunomagnetic concentration and separation assay was developed and applied to PIF for the detection of Cronobacter species. The detection limits of the developed assay were 5.9 × 103 ± 0.7-4.8 × 104 ± 0.2 CFU/mL for Cronobacter species in pure culture with no cross-reactivity with 13 other tested non-Cronobacter strains. Additionally, the developed assay could provide results in 3 h when the contaminated level was higher than 104 CFU/25 g PIF and in 9 h when the contaminated level was 10 CFU/25 g PIF. The developed immunoliposome-based immunomagnetic concentration and separation assay is rapid, sensitive, and simple and thus has great potential for use in the detection of Cronobacter species in PIF.

Development of Fe3O4/Ag core/shell-based multifunctional immunomagnetic nanoparticles for isolation and detection of CD34+ stem cells.

Fe3O4/Ag core/shell nanoparticles functionalized with the free amino (NH2) functional groups (Fe3O4/Ag-NH2) were conjugated with fluorescent electron coupled dye (ECD)-antiCD34 antibody using the 1-ethyl-3-(3'-dimethyl-aminopropyl) carbodiimide (EDC) catalyst (ECD - Electron Coupled Dye or R Phycoerythrin-Texas Red is a fluorescent organic dye attached to the antibody). The characteristic fluorescence of ECD in the antibody was investigated and was used as a good indicator for estimating the percentage of the antibodies that were successfully conjugated with the nanoparticles. The conjugation efficiency was found to increase depending on the VNP:VAB ratio, where VNP and VAB are the volumes of the nanoparticle solution (concentration of 50 ppm) and the as-purchased antibody solution, respectively. The conjugation efficiency rapidly increased from approximately 18% to approximately 70% when VNP:VAB was increased from 2:1 to 100:1, and it gradually reached the saturated state at an efficiency of 95%, as the VNP:VAB was equal to 300:1. The bioactivity of the abovementioned conjugation product (denoted by Fe3O4/Ag-antiCD34) was evaluated in an experiment for the collection of stem cells from bone marrow samples.

Immuno-affinity Amperometric Detection of Bacterial Infections.

A combination of an immuno-affinity enrichment strategy and sensitive amperometric read-out was implemented in a point-of-care platform intended for bacterial infection analysis. Bacterial cells, selectively captured and enriched from complex matrices through immuno-affinity, were detected by amperometric monitoring of the redox state of metabolic activity indicators, providing species identification and viable-cell quantification. The method was successfully employed for the diagnosis of bacterial infections including antimicrobial susceptibility testing with only several hours of total working time.