Sensitive Sandwich Research Articles

Enzyme-linked aptamer-based sandwich assay (ELASA) for detecting Plasmodium falciparum lactate dehydrogenase, a malarial biomarker.

Herein, we report a sensitive and selective enzyme-linked aptamer-based sandwich assay (ELASA) to detect Plasmodium falciparum lactate dehydrogenase (PfLDH), which is an attractive biomarker for malaria diagnosis and antimalarial medication. We performed the sandwich assay with a single aptamer sequence, called 2008s, owing to the structural properties of the PfLDH tetramer instead of using a conventional sandwich assay with two different aptamers (or antibodies) for capturing and probing a target molecule. First, the biotinylated PfLDH aptamer was linked with immobilized streptavidin on a microwell plate for binding flexibility, and then PfLDH was bound to the aptamer. Next, a horseradish peroxidase-conjugated aptamer of the same sequence was used to analyze PfLDH quantitatively. Using this approach, the limit of detection (LOD) of PfLDH with the naked eye was 100 ng mL-1, and the LOD and limit of quantification from the absorbance measurements were 34.9 ng mL-1 and 95.5 ng mL-1, respectively, based on PfLDH spiked blood samples. Our proposed method selectively binds PfLDH, not human lactate dehydrogenase. Therefore, this method may be a valuable tool for diagnosing, monitoring, and quarantining malaria cases easily and rapidly.

An ultrasensitive sandwich chemiluminescent enzyme immunoassay based on phage-mediated double-nanobody for detection of Salmonella Typhimurium in food

In this study, we demonstrated the first report of a phage-mediated double-nanobody sandwich chemiluminescent enzyme immmunoassay (P-CLISA) for Salmonella Typhimurium (S. Typhimurium) determination. Epitope mapping was first conducted for pairing nanobodies, and then soluble and phage-displayed nanobodies were used as detection antibodies in Nb-ELISA and P-ELISA, respectively. Compared with Nb-ELISA, P-ELISA has a 100-fold improvement in sensitivity given the signal amplification mediated by phage. Moreover, a chemiluminescence reaction was applied to replace the traditional chromogenic reaction in the assay, which achieved the detection of S. Typhimurium with an LOD of 3.63 × 103 CFU/mL in a linear range from 5.1 × 103 to 1.2 × 106 CFU/mL. The P-CLISA was successfully applied in actual sample analysis and able to detect fewer than 10 S. Typhimurium cells within 6–8 h of incubation. This study provides guidelines to develop reliable and sensitive double nanobody sandwich immunoassays, which also have great potential for other large molecule monitoring in food samples.

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange–Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

Development of a sensitive sandwich enzyme-linked immunosorbent assay test kit for reliable detection of peanut residues in processed food

Development of a sensitive sandwich enzyme-linked immunosorbent assay test kit for reliable detection of peanut residues in processed food

Association between Leptin and Adiponectin in women with Polycystic Ovary Syndrome in North Indian Population

Polycystic ovary syndrome (PCOS) is one of the most common endocrine abnormalities, characterized by biochemical hyperandrogenaemia, chronic anovulation, and polycystic ovaries. The objective of the present study to determine the association of adiponectin and leptin with women with PCOS in North Indian Women. In the hospital-based case-control study 100 women with PCOS and 100 controls were recuited from RAMA Medical College, Kanpur, India. Five ml venous blood sample was collected under aseptic precaution and transferred in the serum separator tubes. Leptin and adiponectin levels were assayed by human sensitive leptin double-antibody sandwich enzyme-linked immunosorbent one-step process assay (QAYEE-BIO life science) according to instructions provided with the kit. In the multivariable logistic regression analysis, leptin was found to be independently associated with 1.14 times higher risk for PCOS (OR 1.14, 95% CI 1.01 to 1.29). Receiver operating curve (ROC) analysis suggested excellent predictive accuracy of the multivariable model with Area under the curve 0.86. In the ROC analysis leptin was associated with 68% area under the curve for predicting PCOS using cut off value of >7.87, (P<0.001, Sensitivity 87.8, Specificity 49.4). Adiponectin level was not significantly associated with predicting PCOS (Area under the curve 0.54, P =0.30, Sensitivity 71.1, specificity 50.5, cut off value <10.2). To conclude we observed that higher leptin levels were independently associated with the risk of PCOS, however adiponectin level was not independently associated with the risk of PCOS.

Highly selective and sensitive sandwich immunosensor platform modified with MUA-capped GNPs for detection of spike Receptor Binding Domain protein: A precious marker of COVID 19 infection

A label-free electrochemical biosensing system as a suitable analysis technique for COVID 19 specific spike receptor-binding domain protein (RBD) was developed with an aim to facilitate the diagnosis of coronavirus. A novel production procedure for the fabrication of gold nanoparticles (GNPs)-capped 11-mercaptoundecanoic acid (MUA) modified bioelectrode was presented and its application potential for RBD biosensing was examined. The bioelectrode fabrication protocol was based on covalent ester linking formation between hydroxylated ITO electrode and GNPs-capped MUA (GNPs@MUA) with carboxyl ends. For this aim, spherical GNPs were prepared and characterized with scanning-transmission electron microscopy (S-TEM), UV–vis, and Raman spectroscopy. The synthesized GNPs were functionalized with MUA yielding Au-S bonds. Then, covalent immobilization of anti-RBD antibodies on the GNPs@MUA was performed with the help of carbodiimide coupling chemistry. The assembly processes of GNPs@MUA, anti-RBD antibodies and RBD antigens were characterized electrochemical, chemical and morphological techniques. GNPs@MUA was used as immobilization environment and provided the most effective surface design for target immunosensor. The resulting immunosensor is further applied to the impedimetric detection of RBD and it displayed a linear response to RBD antigen in the linear range of 0.002−100 pg mL−1 with a limit of detection of 0.577 fg mL−1 and sensitivity of 0.238 kohmpgmL−1 cm−2. The fabricated immunosensor had a good repeatability, long storage, stability and a reusable property after simple regeneration process. Furthermore, it was successfully employed for selective determination of RBD in artificial nasal secretion samples.

Development of a rapid and sensitive quantum dot nanobead-based double-antigen sandwich lateral flow immunoassay and its clinical performance for the detection of SARS-CoV-2 total antibodies

Owing to the over-increasing demands in resisting and managing the coronavirus disease 2019 (COVID-19) pandemic, development of rapid, highly sensitive, accurate, and versatile tools for monitoring total antibody concentrations at the population level has been evolved as an urgent challenge on measuring the fatality rate, tracking the changes in incidence and prevalence, comprehending medical sequelae after recovery, as well as characterizing seroprevalence and vaccine coverage. To this end, herein we prepared highly luminescent quantum dot nanobeads (QBs) by embedding numerous quantum dots into polymer matrix, and then applied it as a signal-amplification label in lateral flow immunoassay (LFIA). After covalently linkage with the expressed recombinant SARS-CoV-2 spike protein (RSSP), the synthesized QBs were used to determine the total antibody levels in sera by virtue of a double-antigen sandwich immunoassay. Under the developed condition, the QB-LFIA can allow the rapid detection of SARS-CoV-2 total antibodies within 15 min with about one order of magnitude improvement in analytical sensitivity compared to conventional gold nanoparticle-based LFIA. In addition, the developed QB-LFIA performed well in clinical study in dynamic monitoring of serum antibody levels in the whole course of SARS-CoV-2 infection. In conclusion, we successfully developed a promising fluorescent immunological sensing tool for characterizing the host immune response to SARS-CoV-2 infection and confirming the acquired immunity to COVID-19 by evaluating the SRAS-CoV-2 total antibody level in the crowd.

Development and characterization of new swine immune reagents to understand immune correlates for vaccines, infection, and biomedical research outcomes

Abstract The USDA-NIFA Swine Immune Toolkit Initiative has a goal to generate priority immune reagents, based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. Our efforts are aimed at expression of soluble proteins and production of panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production. Generation of new panels of mAbs reactive with porcine IL-5 and IL-21 have been initiated. Panels of mAbs to IL-6, IL-13, IL-17A, IL-28B, CXCL10, and BAFF are being screened for their reactivity in multiple immune assays. Reactivity tests of labeled anti-IL-6, -IL-17A, -IL-13 and -CXCL10 mAbs for intracellular staining of porcine myeloid and T cells using flow cytometry-based assays are in progress. Sensitive sandwich ELISA assays are now available for IL-17A, IL-13 and CXCL10; other targets are being screened for best mAb pairs for such assays. Planning for the generation of SLA-I & -II tetramers to identify swine CD4 and CD8 T cells specific for influenza virus peptides has been initiated. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized techniques using these reagents for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease, vaccine, and biomedical research efforts.

A sensitive sandwich ELISA using a modified biotin-streptavidin amplified system for histamine detection in fish, prawn and crab

Histamine poisoning from seafood is a significant public health and safety concern. To detect histamine sensitively and accurately, a novel competitive sandwich immunoassay using a modified biotin-streptavidin system coupling with polylysine was developed. Using this strategy, a sandwich ELISA with an IC50 value of 112.8 ng mL−1 and a broad linear range of 11.7–1500 ng mL−1 with a correlation coefficient of 0.9942 was validated. Without any sample derivatization procedure, the recovery of histamine ranged from 80.19% to 108.3% with a coefficient of variation of 1.43–11.7% in tuna, prawn and crab. The sandwich ELISA had a detectionlimit of 5.86 ng mL−1, which was 15-fold lower than an indirect competitive ELISA (ic-ELISA). This simple, sensitive and accurate method can be applied to detect histamine in routine seafood samples.

Utility of procalcitonin as an early diagnostic marker of bacteremia in individuals with periodontitis Stage II and III.

The aim of the present study is to assess the ability of procalcitonin (PCT) to differentiate between periodontal health and Stage II and III periodontitis. We further assessed, if PCT can reflect early bacteremia induced by non-surgical periodontal treatment (NSPT). Sixty-four systemically healthy individuals were divided into Group I, periodontally healthy, and Group II, Stage II and III periodontitis. NSPT was done for both the groups. Standardized serum and salivary samples were obtained and analyzed for PCT levels using highly sensitive double antibody sandwich enzyme-linked immunosorbent assay at baseline and 2 weeks. In addition, the serum levels of PCT were recorded at immediate and 1-hour post-NSPT. Mean PCT levels (saliva=0.03ng/mL and serum=0.05ng/mL) in periodontally healthy group were considerably lower than that in the periodontitis group (saliva=0.22ng/mL and serum=1.85ng/mL) with significant intergroup comparison at P< 0.001. Post NSPT the mean serum PCT values increased from 1.854ng/mL to 1.871ng/mL at the immediate interval and remained at 0.879ng/mL after 2 weeks at P< 0.001. Spearman correlation showed highly significant positive correlation between serum and salivary PCT values to clinical attachment level (CAL) at P< 0.001 and rho=0.78 and 0.75, respectively. Linear regression model showed serum PCT to be a significant predictor for CAL. Screening for serum PCT levels in patients with periodontitis could act not only as a guide to assess the bacterial load and use of antibiotics but also as a predictor for CAL loss in patients with periodontitis.

Microparticle Encapsulation of a Prostate-targeted Biologic for the Treatment of Liver Metastases in a Preclinical Model of Castration-resistant Prostate Cancer

PRX302 is a highly potent, mutant bacterial pore-forming biologic protoxin engineered for selective activation by PSA, a serine protease expressed by benign and malignant prostate epithelial cells. Although being developed as a local therapy for benign prostatic hyperplasia and localized prostate cancer, PRX302 cannot be administered systemically as a treatment for metastatic disease due to binding to ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored proteins, which leads to poor accumulation within the tumor microenvironment. To overcome this limitation, poly-lactic-co-glycolic acid (PLGA) microparticles encapsulating the protoxin were developed, which are known to accumulate in the liver, a major site of metastasis for prostate cancer and other solid tumors. A highly sensitive and reproducible sandwich ELISA to quantify PRX302 released from microparticles was developed. Utilizing this assay, PRX302 release from different microparticle formulations was assessed over multiple days. Hemolysis assays documented PSA-dependent pore formation and lytic potential (i.e., function) of the released protoxin. MTT assays demonstrated that conditioned supernatant from PRX302-loaded, but not blank (i.e., unloaded), PLGA microparticles was highly cytotoxic to PC3 and DU145 human prostate cancer cells in the presence of exogenous PSA. Microparticle encapsulation prevented PRX302 from immediately interacting with GPI-anchored proteins as demonstrated in a competition assay, which resulted in an increased therapeutic index and significant antitumor efficacy following a single dose of PRX302-loaded microparticles in a preclinical model of prostate cancer liver metastasis with no obvious toxicity. These results document that PRX302 released from PLGA microparticles demonstrate in vivo antitumor efficacy in a clinically relevant preclinical model of metastatic prostate cancer.

Association of LPL Gene Variant and Serum LPL Level with Ischemic Stroke in Iraqi Population

Background: Lipoprotein lipase (LPL) plays an important role in plasma lipoprotein metabolism.An increasing number of studies have suggested an association of LPL gene variants with the risk ofcardiovascular and cerebrovascular diseases.Objective: To analyze whether Hind III polymorphism of LPL gene and serum LPL level are associatedwith ischemic stroke in Iraqi population.Method: Fifty ischemic stroke patients (clinical diagnosis and x-ray CT) and fifty controls were enrolled inthis case–control study. The LPL Hind III polymorphism was determined by PCR-RFLP technique and LPLmass level was estimated using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA).Results: In the present research was not found any association between the Hindlll LPL gene polymorphismand acute ischemic stroke in the population studied; the allele and genotypic frequencies of the studiedpolymorphism was similar in cases and controls and followed the Hardy-Weinberg equilibrium. Therelationship between Hind III genotypes and the LPL mass level was analyzed using ANOVA and furtherconfirmed by Post-hoc analysis. there was no significant difference in LPL mass levels between the genotypegroups H+H+, H+H-, and H-H- (p value&gt;0.05).Conclusions: The Hind III polymorphism of LPLis not a genetic marker for the development of ischemicstroke as well as not determinants of serum LPL level in the Iraqian sample used.

Convenient Construction of Orthogonal Dual Aptamer-Based Plasmonic Immunosandwich Assay for Probing Protein Disease Markers in Complex Samples and Living Animals.

Aptamers, because of their outstanding merits including simple synthesis and easy modification, have been widely used as antibody alternatives to construct novel immunosandwich assays. Dual aptamer-based sandwich assays exhibit multiple advantages over conventional immunosandwich assays and single aptamer-based sandwich assays. However, their construction is hampered by the limited knowledge of binding orthogonality of aptamers reported in the literature. Herein, we present a new strategy for conveniently constructing an orthogonal dual aptamer-based plasmonic immunosandwich assay (odA-PISA) for probing proteins in complex samples and living animals. An orthogonal aptamer pair was first efficiently selected from the aptamers reported in the literature by affinity capillary electrophoresis. Then, a target protein-capturing gold thin-layer-coated probe and silver nanoparticle-based Raman labeling nanotags were conveniently prepared with the selected aptamers and used to construct the assay. The double aptamers used ensured the specificity, whereas the plasmonic coupling effect between the target-capturing probe and the generated Raman nanotags significantly enhanced the Raman signal intensity, providing high sensitivity. As a proof of principle, alkaline phosphatase (ALP) was used as the target. The constructed odA-PISA exhibited high specificity and high sensitivity toward ALP, giving cross-reactivity ≤ 4.2% and the limit of detection of 3.8 pM (S/N = 4). The quantitative determination of ALP in human serum and probing ALP in tumor-bearing mice were achieved, showing the great application potential of the method. This strategy is widely applicable to other protein disease markers. Therefore, it opened a new access to the construction of sensitive dual aptamer-based sandwich assays for real-world applications, particularly disease diagnosis.

Quantitative pharmacokinetic evaluation of Subtilisin QK-2 after a bolus IV injection in a rat model using a novel sandwich enzyme-linked immunosorbent assay

Intravascular thrombosis is a main cause of multiple cardiovascular diseases. A high thrombolytic activity of the microbial fibrinolytic enzyme Subtilisin QK-2, which is highly homologous to Nattokinase, shows great exploitable potential in thrombolytic therapy. However, the lack of a sensitive detection method limits the further analysis of Subtilisin QK-2 in vivo. We prepared a polyclonal antibody and four monoclonal antibodies (IgG1, titers of 1:500,000) to establish a sensitive sandwich ELISA for Subtilisin QK-2 detection. The limit of detection (LOD) of this ELISA was 1.160 ng/mL. The linear range of the standard curve was 1.96−250 ng/mL (R2 = 0.9912). The cut-off value was 0.236. Subsequently, a pharmacokinetic dose (IV bolus) was administered and analyzed with the established ELISA. The concentration-time profiles were best fitted to a two-compartment model. T1/2α values for doses of 2 mg/kg, 4 mg/kg, and 8 mg/kg were 29.90 ± 10.02 min, 27.17 ± 1.96 min, and 21.83 ± 9.95 min, and T1/2β values were 144.43 ± 49.73 min, 173.46 ± 52.58 min, and 159.49 ± 48.75 min, respectively. Subtilisin QK-2 was eliminated through a mechanism with first-order kinetics. In conclusion, this study provides useful data for further research and clinical applications of Subtilisin QK-2 in the treatment of cardiovascular diseases.

Development of a sensitive monoclonal antibody-based sandwich ELISA to detect Vip3Aa in genetically modified crops

ObjectivesTo develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to detect Vip3Aa in genetically modified (GM) crops and their products.ResultsVegetative insecticidal proteins (Vips) are secreted by Bacillus thuringiensis (Bt) and are known to be toxic to Lepidoptera species. Vip3Aa family proteins, Vip3Aa19 and Vip3Aa20, were successfully applied in GM crops to confer an effective and persistent insecticidal resistance. A sensitive monoclonal antibody-based sandwich ELISA was developed to detect Vip3Aa in GM crops and their products. Two monoclonal antibodies were raised against the overexpressed and purified His-Vip3Aa20, were purified from mouse ascites and characterized. A sandwich ELISA method was developed using the 2G3-1D7 monoclonal antibody for capture and the biotin-labeled 1F9-1F5 monoclonal antibody for detection of Vip3Aa20. The linear detection range of the method was found to be approximately 31.25–500 pg/ml, with a sensitivity of 10.24 pg/ml.ConclusionsThe established ELISA was effective for detecting Vip3Aa family proteins other than Vip3Aa8, and was successfully applied in the detection of Vip3Aa20 and Vip3Aa19 expressed in transgenic maize and cotton.

Gold nanoparticles enumeration with dark-field optical microscope for the sensitive glycoprotein sandwich assay

Protein glycosylation is an important post-translational modification and glycoproteins are associated with many crucial metabolic progresses of life. In order to detect glycoproteins sensitively, we propose a gold nanoparticles (GNPs) enumeration method based on boronate affinity sandwich system, which is constructed between the boronic acid polymer functionalized magnetic nanoparticles (Fe3O4@MPS@VPBA NPs) and 4-mercaptophenylboronic acid modified GNPs (GNPs-MPBA) by the targeted glycoproteins as the linker. Therefore, the sandwich complex is formed, resulting in the decrease of GNPs-MPBA counts in the solution. Based on the dark-field microscope (DFM) imaging technique, the sensitive GNPs enumeration assay is developed for glycoproteins quantitation. Immunoglobulin (IgG), as one of the important glycoproteins, is introduced to evaluate the proposed method. A low detection limit of 1.22 ng mL−1 for IgG analysis is obtained. The result indicates that the proposed GNPs enumeration method offers a simple, effective, label-free and highly sensitive strategy without signal amplification. It also possesses great potential for various target molecules determination at the single-particle level in the future.

Highly sensitive sandwich electrochemical sensor based on DNA-scaffolded bivalent split aptamer signal probe

Split aptamers find growing interest as recognition elements for sandwich electrochemical sensors. Here, we report a convenient and highly sensitive sandwich electrochemical sensor based on a new conception of bivalent split aptamer signal probe, which is readily engineered through a simple hybridization of two identical monovalent split aptamers to a DNA template strand. Because of the bivalence cooperativity enhanced sandwich binding to target molecules, this type of DNA-scaffolded bivalent signal probe exhibits significantly increased signal gain for sandwich electrochemical detection of target analytes in comparison with the conventional monovalent signal probe. Through rational optimization of the length of anchor duplex in DNA scaffold and concentration of bivalent signal probe in electrochemical test solution, high signal sensitivity for ATP detection is achieved with a detection limit of 41 nM. The DNA-scaffolded bivalent signal probe based sandwich electrochemical sensor is also specific, selective and applicable for ATP detection in real samples. Due to its simplicity in design and high sensitivity for target recognition, we hope that the conception of DNA-scaffolded bivalent signal probe will provide a useful guide to further developing split aptamer-based sandwich electrochemical sensors for a wide variety of other target analytes.

Fabrication of Fe3O4/Au@ATP@Ag Nanorod sandwich structure for sensitive SERS quantitative detection of histamine

We have successfully prepared a highly sensitive sandwich nanosensor combined Fe3O4 and Au@ATP@Ag nanorods for histamine detection based on surface-enhanced Raman spectroscopy (SERS). The Fe3O4 beads with –COOH served as a capture part to enrich histamine. The Au@ATP@Ag core-shell nanorods functionalized with Nalpha,Nalpha-Bis(carboxymethyl)-l-lysine (AB-NTA) were then used to connect with the imidazolyl group of histamine, simultaneously the internal standard 4-aminothiophenol (4-ATP) in the core-shell structure was used as the SERS signal. PLS regression model based on concentration range 10−3-10−8mol/L showed a linear trend with R2 = 0.9907. Our new approach can quickly and reliably determine histamine in fish sample and RAW264.7 cell lysates. This protocol for histamine extraction and SERS analysis enables the development of ultra-sensitive method for histamine detection.

Erythroferrone as a sensitive biomarker to detect stimulation of erythropoiesis.

Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to erythropoietin stimulation. ERFE suppresses the hepatic synthesis of the master iron-regulatory hormone, hepcidin. The impact of erythropoiesis stimulation on ERFE secretion in humans is poorly understood. This paucity of information is due in part to the lack of available means for ERFE quantification in serum samples. The present study tested a new sensitive sandwich immunoassay for human ERFE. This assay was used to demonstrate that injection of various erythropoiesis stimulating agents (ESAs) increased the blood ERFE levels in healthy volunteers. After exogenous stimulation of erythropoiesis, ERFE increased up to 8-fold with a detection window of 13 days. The impact of one unit of blood withdrawal on erythropoiesis stimulation of ERFE was also tested. ERFE significantly increased after blood withdrawal in subjects injected with both iron and saline solution, suggesting that iron supplementation did not mask the ERFE increase after blood withdrawal. The effects of exercise-induced muscle damage on ERFE was assessed by comparing ERFE levels with creatine kinase levels in samples from subjects with heavy exercise loads, and determined that this was not a confounder. The ERFE assay is a sensitive means to investigate the connection between iron metabolism and erythropoiesis in humans, and to detect ESA abuse in the antidoping field.

A sensitive SERS-based sandwich immunoassay platform for simultaneous multiple detection of foodborne pathogens without interference.

A reliable and sensitive sensing of multiple foodborne pathogens is critical for timely diagnosis and human health. To meet this need, herein, we designed a sandwich immunoassay platform, using functionalized SERS probes and magnetic beads, for the interference-free simultaneous detection of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in food samples by surface-enhanced Raman scattering (SERS) technology. The signal of two SERS probes coded by triple bonds (C[triple bond, length as m-dash]C and C[triple bond, length as m-dash]N) located at 2105 and 2227 cm-1, respectively, could perfectly avoid the spectral overlap with coexisting materials in the Raman fingerprint region, which ensured the accuracy of the immunoassay platform. The application of functional magnetic beads, integrating enrichment and separation, greatly improved the sensitivity of the detection system. Under magnetic force, due to the mature interaction between the antigen and antibody, the sandwich immunoassay platform could be fabricated. Its limit of detection (LOD) for the simultaneous detection of E. coli and S. aureus was as low as 10 and 25 cfu mL-1, respectively, and the sandwich immunoassay platform was successfully applied for the detection of E. coli and S. aureus in bottled water and milk. As a sensitive and highly selective analytical technique for the simultaneous multiple detection of pathogens, this SERS-based method has great potential to be applied in the field of food safety.