Fluorescence Sandwich Immunoassay Research Articles

A novel nanoparticle-based fluorescent sandwich immunoassay for specific detection of Salmonella Typhimurium

The diseases caused by foodborne pathogens have a serious impact on human health and social stability. Conventional detection methods can involve long assay times and complex pretreatment steps, making them unsuitable for rapid, large-scale analysis of food samples. We constructed a novel nano-fluorescence sandwich immunosorbent immunoassay (nano-FSIA) to rapidly detect Salmonella Typhimurium in food, based on strong covalent binding between streptavidin and biotin. We used antibodies coupled to large particle-size fluorescent microspheres as fluorescent probes for direct quantitative analysis of S. typhimurium in milk. The optimized parameters were determined, and specificity and sensitivity were validated in phosphate-buffered saline (PBS) and milk. The results demonstrated a wide dynamic detection range for S. typhimurium (103–108 colony forming units [CFU]/mL), with the limit of detection in PBS and milk at 234 and 346 CFU/mL, respectively. The results of nano-FSIA were consistent with those of plate counts and enzyme-linked immunosorbent assays, providing an effective and promising single-bacterium counting method for the rapid detection of Salmonella.

On-Chip Analysis of Protein Secretion from Single Cells Using Microbead Biosensors.

The profiling of the effector functions of single immune cells─including cytokine secretion─can lead to a deeper understanding of how the immune system operates and to potential diagnostics and therapeutical applications. Here, we report a microfluidic device that pairs single cells and antibody-functionalized microbeads in hydrodynamic traps to quantitate cytokine secretion. The device contains 1008 microchambers, each with a volume of ∼500 pL, divided into six different sections individually addressed to deliver an equal number of chemical stimuli. Integrating microvalves allowed us to isolate cell/bead pairs, preventing cross-contamination with factors secreted by adjacent cells. We implemented a fluorescence sandwich immunoassay on the biosensing microbeads with a limit of detection of 9 pg/mL and were able to detect interleukin-8 (IL-8) secreted by single blood-derived human monocytes in response to different concentrations of LPS. Finally, our platform allowed us to observe a significant decrease in the number of IL-8-secreting monocytes when paracrine signaling becomes disrupted. Overall, our platform could have a variety of applications for which the analysis of cellular function heterogeneity is necessary, such as cancer research, antibody discovery, or rare cell screening.

Supersensitive detection of single-histamine molecule on nanoplates by turn-on small molecule fluorescence sandwich immunoassay.

We develop a supersensitive "turn-on format" fluorescence sandwich immunoassay for detecting small single molecules. Gold nanoplate-based biotin antibodies and streptavidin-fluorophores were used instead of streptavidin-horseradish peroxidase reacting with a biotin tracer in a microplate-based competitive enzyme-linked immunosorbent assay (ELISA). Our platform showed a low detection limit of 5 zeptomolar (5 × 10-21 M), 5.4 × 1010 times higher detection sensitivity than the conventional tune-off format ELISA.

Development of cellulosic material-based microchannel device capable of fluorescence immunoassay of microsamples.

Microfluidic immunoassay devices are a promising technology that can quickly detect biomarkers with high sensitivity. Recently, many studies implementing this technology on paper substrates have been proposed for improving cost and user-friendliness. However, these studies have identified problems with the large volume of sample required, low sensitivity, and a lack of quantitative accuracy and precision. In this paper, we report a novel structure implemented as a cellulosic material-based microchannel device capable of quantitative immunoassay using small sample volumes. We fabricated microfluidic channels between a transparent cellophane film and water-resistant paper to facilitate loading of small-volume samples and reagents, with a 40-μm-wide immunoreaction matrix constructed in the center of the microchannel using highly precise photolithography. A fluorescence sandwich immunoassay for C-reactive protein (CRP) was successfully implemented that required only a 1-μL sample volume and a 20-min reaction time. We confirmed that the limit of detection of the device was 10-20 ng/mL with a coefficient of variation under 5.6%, which is a performance level comparable to conventional plastic-based human CRP enzyme-linked immunosorbent assay (ELISA) kits. We expect that such devices will lead to the elimination of large amounts of medical waste from the use of ubiquitous diagnostics, a result that is consistent with environmental sustainability goals.

A fluorescence sandwich immunoassay for the real-time continuous detection of glucose and insulin in live animals.

Real-time biosensors that can continuously measure circulating biomolecules in vivo would provide valuable insights into a patient’s health status and their response to therapeutics, even when there is considerable variability in pharmacokinetics and pharmacodynamics across patient populations. Unfortunately, current real-time biosensors are limited to a handful of analytes (e.g. glucose and blood oxygen) and are limited in sensitivity (high nanomolar). In this work, we describe a general approach for continuously and simultaneously measuring multiple analytes with picomolar sensitivity and sub-second temporal resolution in a device with an incubation time of 30 s. As exemplars, we report the simultaneous detection of glucose and insulin in live diabetic rats. Using our system, we demonstrate the capacity to resolve inter-individual differences in the pharmacokinetic response to insulin, and discriminate profiles from different insulin formulations at high temporal resolution. Critically, our approach is general and could be readily modified to continuously and simultaneously measure other circulating analytes in vivo, thus making it a versatile tool for biomedical research.

A Multiplex Immunosensor for Detecting Perchlorate-Reducing Bacteria for Environmental Monitoring and Planetary Exploration.

Perchlorate anions are produced by chemical industries and are important contaminants in certain natural ecosystems. Perchlorate also occurs in some natural and uncontaminated environments such as the Atacama Desert, the high Arctic or the Antarctic Dry Valleys, and is especially abundant on the surface of Mars. As some bacterial strains are capable of using perchlorate as an electron acceptor under anaerobic conditions, their detection is relevant for environmental monitoring on Earth as well as for the search for life on Mars. We have developed an antibody microarray with 20 polyclonal antibodies to detect perchlorate-reducing bacteria (PRB) strains and two crucial and highly conserved enzymes involved in perchlorate respiration: perchlorate reductase and chlorite dismutase. We determined the cross-reactivity, the working concentration, and the limit of detection of each antibody individually and in a multiplex format by Fluorescent Sandwich Microarray Immunoassay. Although most of them exhibited relatively high sensitivity and specificity, we applied a deconvolution method based on graph theory to discriminate between specific signals and cross-reactions from related microorganisms. We validated the system by analyzing multiple bacterial isolates, crude extracts from contaminated reactors and salt-rich natural samples from the high Arctic. The PRB detecting chip (PRBCHIP) allowed us to detect and classify environmental isolates as well as to detect similar strains by using crude extracts obtained from 0.5 g even from soils with low organic-matter levels (<103 cells/g of soil). Our results demonstrated that PRBCHIP is a valuable tool for sensitive and reliable detection of perchlorate-reducing bacteria for research purposes, environmental monitoring and planetary exploration.

A fluorescence immunoassay for a rapid detection of Listeria monocytogenes on working surfaces

Listeria monocytogenes is a foodborne pathogen responsible for human listeriosis. The increasing incidence of listeriosis induced governments and food manufacturing enterprises to act to diminish the problem. Several methods for the detection of Listeria monocytogenes in food industries were developed. However, they are time-consuming and require the use of specialized equipment. To reduce the detection time of Listeria monocytogenes in food, in this work we developed a fluorescence sandwich immunoassay based on the use of an innovative chitosan-cellulose nanocrystal (CNC) membrane that improves the antigen capture during bacterial growth. The combined use of CNC film for the capture of p60 protein-specific antigen together with the use of fluorescence detection reduced the time of analysis from 24 to 12 h with a limit of detection (LOD) of the assay of 102 CFU/mL (2 Log). In addition, the use of monoclonal anti-PepD covalently immobilized to a CNC membrane assured a high specificity of the assay. Interestingly, the obtained results show no cross-reactivity with the five most diffused pathogen bacteria strains tested.

Microfluidic immunoassay for point-of-care testing using simple fluid vent control

Microfluidic immunoassays have gained immense importance as a point-of-care testing (POCT) platform owing to their low reagent volume requirements, low cost, portability, and rapid reaction. However, they are characterized by inconsistency and lack of reproducibility. In this study, we developed a microfluidic immunoassay platform with a simple signal enhancement technique using fluid vent control. The components of this fluorescence-based immunoassay were successfully pre-loaded in the microfluidic device. The analytes reacted with the detection antibody conjugated on fluorescence beads, and bound to capture the antibody immobilized zone during channel flow. The fluid vent was closed using a small PC-connected equipment when the fluid reached the target displacement. The pausing of the fluid provided sufficient time for the immune reaction, and this addition of the fluid pausing significantly enhanced the immune reaction and increased the limit of detection (LOD) of the immunoassay. The LOD of the developed assay was the same as that of the sandwich fluorescence immunoassay in terms of quantity. Furthermore, the LOD was significantly higher than that of the commercial rapid kits. Therefore, our simple technique could be employed for cost-effective and accurate immunoassays in the field of POCT.

Integrated Microfluidic Device for Functional Secretory Immunophenotyping of Immune Cells.

Integrated platforms for automatic assessment of cellular functional secretory immunophenotyping could have a widespread use in the diagnosis, real-time monitoring, and therapy evaluation of several pathologies. We present a microfluidic platform with integrated biosensors and culture chambers to measure cytokine secretion from a consistent and uniform number of immune cells. The biosensor relies on a fluorescence sandwich immunoassay enabled by the mechanically induced trapping of molecular interactions method. The platform contains 32 cell culture chambers, each patterned with an array of 492 microwells, to capture and analyze both adherent and nonadherent immune cells. Multiple stimuli can be delivered to a set of culture chambers. Per chamber, we were able to capture consistently 1113 ± 191 of blood-derived monocytes and neutrophils and 348 ± 37 THP-1 monocytes. Good occupancy efficiencies of ∼70% with a uniformity of ∼90% across all of the culture chambers of the device were achieved. Furthermore, we demonstrate that up to 96% of cells remain viable for the first 48 h. The employment of epoxy-modified glass substrates and active mixing enhanced the biosensing performance compared to the use of bare glass and simple diffusion. Finally, we performed functional secretory analysis of interleukin-8 and tumor necrosis factor alpha from human neutrophils and monocytes, stimulated with various doses of lipopolysaccharide and phorbol 12-myristate 13-acetate-ionomycin, respectively. We foresee the employment of our microfluidic platform in the diagnosis of different pathologies where alterations in cytokine secretion patterns can be used as biomarkers.

In Pursuit of Zero 2.0: Recent Developments in Nonfouling Polymer Brushes for Immunoassays.

"Nonfouling" polymer brush surfaces can greatly improve the performance of in vitro diagnostic (IVD) assays due to the reduction of nonspecific protein adsorption and consequent improvement of signal-to-noise ratios. The development of synthetic polymer brush architectures that suppress adventitious protein adsorption is reviewed, and their integration into surface plasmon resonance and fluorescent sandwich immunoassay formats is discussed. Also, highlighted is a novel, self-contained immunoassay platform (the D4 assay) that transforms time-consuming laboratory-based assays into a user-friendly and point-of-care format with a sensitivity and specificity comparable or better than standard enzyme-linked immunosorbent assay (ELISA) directly from unprocessed samples. These advancements clearly demonstrate the utility of nonfouling polymer brushes as a substrate for ultrasensitive and robust diagnostic assays that may be suitable for clinical testing, in field and laboratory settings.

Microfluidic smartphone quantitation of Escherichia coli in synthetic urine

In spite of the clinical need, there is a major gap in rapid diagnostics for identification and quantitation of E. coli and other pathogens, also regarded as the biggest bottleneck in the fight against the spread of antimicrobial resistant bacterial strains. This study reports for the first time an optical, smartphone-based microfluidic fluorescence sandwich immunoassay capable of quantifying E. coli in buffer and synthetic urine in less than 25 min without sample preparation nor concentration. A limit of detection (LoD) up to 240 CFU/mL, comensurate with cut-off for UTIs (103–105 CFUs/mL) was achieved. Replicas of full response curves performed with 100–107 CFUs/mL of E. coli K12 in synthetic urine yielded recovery values in the range 80–120%, assay reproducibility below 30% and precision below 20%, therefore similar to high-performance automated immunoassays. The unrivalled LoD was mainly linked to the ‘open fluidics’ nature of the 10-bore microfluidic strips used that enabled passing a large volume of sample through the microcapillaries coated with capture antibody. The new smartphone based test has the potential of being as a rapid, point-of-care test for rule-in of E. coli infections that are responsible for around 80% of UTIs, helping to stop the over-prescription of antibiotics and the monitoring of patients with other symptomatic communicable diseases caused by E. coli at global scale.

A tyrosinase-induced fluorescence immunoassay for detection of tau protein using dopamine-functionalized CuInS2/ZnS quantum dots.

Rapid, highly sensitive detection of tau protein and other neurodegenerative biomarkers remains a significant hurdle for diagnostic tests for Alzheimer's disease. In this work, we developed a novel tyrosinase (TYR)-induced tau aptamer-tau-tau antibody (anti-tau) sandwich fluorescence immunoassay to detect tau protein that used dopamine (DA)-functionalized CuInS2/ZnS quantum dots as the fluorophore. CuInS2/ZnS core/shell quantum dots with high luminescence, low toxicity, and excellent biocompatibility were successfully fabricated and decorated with DA through amide conjugation. Meanwhile, TYR was conjugated with anti-tau by a click reaction. When DA-functionalized CuInS2/ZnS quantum dots were added to the sandwich system, TYR catalyzed the transformation of DA to dopamine quinone, which acted as an effective electron acceptor and triggered fluorescence quenching. The fluorescence intensity of the immunoassay based on DA-functionalized CuInS2/ZnS quantum dots shows good performance in terms of linearity with the logarithm of tau protein concentration, with a linear concentration range from 10 pM to 200 nM. This work is the first to use a TYR-induced fluorescence immunoassay for the rapid detection of tau protein, paving a new way for the detection of disease biomarkers. Graphical abstract.

Microfluidic device with a push–pull sequential solution-exchange function for affinity sensing

A simple valveless microfluidic device consisting of a reaction chamber and eight flow channels was fabricated for the detection of proteins. The efficient exchange of solutions in the reaction chamber was achieved by sequentially introducing solutions into the reaction chamber and removing them from the reaction chamber through the same flow channels. The influence of the hydrophobicity of the reaction chamber and the viscosity of a blocking solution on solution exchange was investigated. The analytical performance of the device was examined by detecting human interleukin 2 (IL-2) using a sandwich fluorescence immunoassay, with the amount of immuno-complex formed in the reaction chamber monitored by fluorescence microscopy. The approach facilitated the rapid detection of IL-2, in which the total assay time was less than 30 min. A clear dependence of fluorescence intensity on IL-2 concentration was observed over the 125 pg/mL–2.0 ng/mL range, with a detection limit of 105 pg/mL. To promote automation, a simple mechanism that exchanges solutions by the push–pull movement of a PDMS diaphragm was also demonstrated.

Plasmonic Hepatitis B Biosensor for the Analysis of Clinical Saliva.

A biosensor for the detection of hepatitis B antibodies in clinical saliva was developed. Compared to conventional analysis of blood serum, it offers the advantage of noninvasive collection of samples. Detection of biomarkers in saliva imposes two major challenges associated with the low analyte concentration and increased surface fouling. The detection of minute amounts of hepatitis B antibodies was performed by plasmonically amplified fluorescence sandwich immunoassay. To have access to specific detection, we prevented the nonspecific adsorption of biomolecules present in saliva by brushes of poly[(N-(2-hydroxypropyl) methacrylamide)-co-(carboxybetaine methacrylamide)] grafted from the gold sensor surface and post modified with hepatitis B surface antigen. Obtained results were validated against the response measured with ELISA at a certified laboratory using serum from the same patients.

Magnetic bead fluorescent immunoassay for the rapid detection of the novel inflammation marker YKL40 at the point-of-care

Pneumonia is one of the leading causes of death worldwide. We present a magnetic bead fluorescent sandwich immunoassay that allows rapid serum measurement of the novel inflammation marker YKL40 (CHI3L1) at the point of care (POC) that could aid pneumonia diagnosis. The magnetic beads serve as the solid phase for separation of YKL40 from serum. The readout is performed using a small and robust fluorescence reader, which detects the turnover of a fluorescent substrate. The assay procedure, from sample addition to data retrieval, consists of three steps and is performed in less than 20min. The presented assay has a linear range from 3 to 111ng/mL, with a limit of detection of 2.9ng/mL. The average recoveries were found between 101 and 111%. The developed method was applied in sera from healthy subjects (n=14; c(YKL40)=50±49ng/mL) and from pneumonia patients (n=14; c(YKL40)=333.6±225ng/mL). The elevated YKL40 concentrations in pneumonia-diseased patients are in good agreement with previously published data. The POC-ready device provides a simple immunoassay that could help to optimize pneumonia inflammation diagnostics in low-resource settings.

Correlations of salivary biomarkers with clinical assessments in patients with cystic fibrosis.

RationaleMonitoring clinical disease status in cystic fibrosis frequently requires invasive collection of clinical samples. Due to its noninvasive collection process and direct anatomic relationship with the lower airway, saliva shows great potential as a biological fluid for cystic fibrosis monitoring.ObjectivesTo measure the levels of multiple protein markers in human saliva supernatants and investigate the possibility of utilizing them to provide a more quantitative measure of disease state for use in research and monitoring of patients with cystic fibrosis clinically.MethodsWhole saliva samples were collected and processed from cystic fibrosis patients at two distinct time points (2010 and 2013) and measured by two separate platforms. In this cross sectional study, a convenience sample of 71 participants were recruited with samples measured by multiplexed fluorescence microarray (fiber microarray) and another 117 participant samples were measured by an automated, point-of-care, analyzer (SDReader) using a microsphere-based array via fluorescence sandwich immunoassay. For comparison, saliva from 56 and 50 healthy subjects were collected, respectively. The levels of six target proteins were quantified. Various demographic and clinical data, including spirometry, medical history, and clinicians’ assessments were also collected from patients with cystic fibrosis on the day of saliva collection.Measurements and Main ResultsSimilar trends were observed with both platforms and compared with healthy subjects, cystic fibrosis patients had significantly elevated levels of VEGF, IP-10, IL-8, and EGF as well as lower levels of MMP-9 (P ≤ 0.005) using fiber microarray and significantly elevated levels of IP-10, IL-8 with lower levels of MMP-9 and IL-1β (P ≤ 0.02) using the SDReader. The levels of the six proteins correlated with each other significantly, and in some cases, biomarker levels could be used to differentiate between subgroups of patients with different clinical presentations. For example, IP-10 levels significantly correlated with FEV1 and disease severity (as evaluated by clinicians) with both platforms (P < 0.05).ConclusionsSignificant variations of the levels of six proteins in saliva supernatants, and the correlations of these levels with clinical assessments, demonstrated the potential of saliva for cystic fibrosis research and monitoring.

Increased B-type natriuretic peptide levels in early-onset versus late-onset preeclampsia

We compared B-type natriuretic peptide (BNP) levels, clinical and laboratory findings in early-onset preeclampsia (EOP), late-onset preeclampsia (LOP) and healthy pregnant groups. We studied 40 healthy pregnant and 40 preeclamptic patients. Preeclamptics were divided in two groups, the EOP group (n=20) and LOP group (n=20), according to gestational age at the onset of disease. The distinction criterion for early- vs. late-onset was set as week 34 of gestation. The concentration of the BNP levels was measured by a sandwich fluorescence immunoassay. For statistical analysis of the clinical and laboratory findings non-parametric methods were applied. BNP levels were higher in EOP [61.35 (36.95-93.25) pg/mL] and LOP patients [32.4 (19.15-39.2) pg/mL] than in healthy pregnant women [10.05 (6.08-16.03) pg/mL] (both p<0.001). Furthermore, EOPs had significantly higher BNP levels as compared to LOP patients (p<0.001). A BNP cut-off <24.5 pg/mL had a negative-predictive value of 85.1% excluding preeclampsia. There was a significant inverse correlation between plasma BNP levels of EOP patients and sodium (p<0.05) and total protein concentrations (p<0.05). In the EOP group, a significant positive correlation was observed between plasma levels of BNP and hematocrit (p<0.05), serum potassium (p<0.05), urea (p<0.05) and 24-h proteinuria (p<0.05). BNP levels were significantly higher in EOP than in LOP patients. The cut-off value <24.5 pg/mL seems to be a powerful discriminative indicator excluding preeclampsia. The amount of proteinuria and total protein levels correlate with the elevation of the BNP levels. In EOP the extent of proteinuria is higher than in the LOP.

Moving the solid phase: a platform technology for cartridge based sandwich immunoassays

We report on a cartridge based platform for complex immunoassay formats that allows for flexible adaption of individual steps. It is a sample-to-answer system which is quantitative as well as sensitive. The target molecules are detected through a magnetic bead-based fluorescence sandwich immunoassay. The beads both constitute the solid phase for immobilizing capture molecules and are used for magnetic field activated incubation. The injection molded cartridge comprises several chambers separated by capillary valves. Chambers contain the assay reagents, through which the beads are manipulated via externally applied magnetic fields. Active incubation is made possible by assembling the beads into microstirrers and systematically scanning through a chamber. The beads are transported by focusing them to form an aggregate which subsequently is dragged through the valves. Once the aggregate enters a chamber, it is re-dispersed and magnetic actuation is used to re-assemble the beads into microstirrers. The assay protocol involves an incubation of sample with antibody coated magnetic beads, followed by steps for washing or separation, labeling with fluorescent detection antibody and finally fluorescence detection. An interleukin-8 assay served as a model for evaluating the system and a concentration as low as 5 pg/mL (0.625 pM) was successfully detected. The platform shows potential to be developed into a diagnostic tool to be used in a point-of-care testing (PoCT) environment.

Fluorescence-based immunoassay for human chorionic gonadotropin based on polyfluorene-coated silica nanoparticles and polyaniline-coated Fe3O4 nanoparticles

We report on an ultrasensitive fluorescence immunoassay for human chorionic gonadotrophin antigen (hCG). It is based on the use of silica nanoparticles coated with a copolymer (prepared from a fluorene, a phenylenediamine, and divinylbenzene; PF@SiO2) that acts as a fluorescent label for the secondary monoclonal antibody to β-hCG antigen. In parallel, Fe3O4 nanoparticles were coated with polyaniline, and these magnetic particles (Fe3O4@PANI) served as a solid support for the primary monoclonal antibody to β-hCG antigen. The PF@SiO2 exhibited strong fluorescence and good dispersibility in water. A fluorescence sandwich immunoassay was developed that enables hCG concentrations to be determined in the 0.01–100 ng·mL−1 concentration range, with a detection limit of 3 pg·mL−1.

Protein Microarrays for Quantitative Detection of PAI-1 in Serum.

Plasminogen activator inhibitor-1 (PAI-1), one crucial component of the plasminogen activator system, is a major player in the pathogenesis of many vascular diseases as well as in cancer. High levels of PAI-1 in breast cancer tissue are associated with poor prognosis. The aim of this study is to evaluate rigorously the potential of serum PAI-1 concentration functioning as a general screening test in diagnostic or prognostic assays. A protein-microarray-based sandwich fluorescence immunoassay (FIA) was developed to detect PAI-1 in serum. Several conditions of this microarray-based FIA were optimized to establish an efficacious method. Serum specimens of 84 healthy women and 285 women with breast cancer were analyzed using the optimized FIA microarray. The median serum PAI-1 level of breast cancer patients was higher than that of healthy women (109.7 ng/ml vs. 63.4 ng/ml). Analysis of covariance revealed that PAI-1 levels of the two groups were significantly different (P<0.001) when controlling for an age effect on PAI-1 levels. However, PAI-1 values in TNM stage I-IV patients respectively were not significantly different from each other. This microarray-based sandwich FIA holds potential for quantitative analysis of tumor markers such as PAI-1.