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Standard curve showing the average signal intensity versus concentration for the myoglobin sandwich immunoassay for four different experiments using the target protein. The inset shows the lower concentrations on the standard curve, error bars represent the standard deviation among data sets. Signal intensity has been normalized to the background intensity (zero concentration) for each data set.
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To achieve improved sensitivity in cardiac biomarker detection, a batch incubation magnetic microbead immunoassay was developed and tested on three separate human protein targets: myoglobin, heart-type fatty acid binding protein, and cardiac troponin I. A sandwich immunoassay was performed in a simple micro-centrifuge tube allowing full dispersal o...
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... experiments exhibit a similar result. Fig. 3 shows the average uorescence intensity of data collected from four separate experiments with independent dilutions of a myoglobin stock sample. Error bars show the standard devia- tion of each data set. Differences in overall uorescence inten- sity were observed between experiments, due to aging of the mercury lamp used to illuminate ...
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Citations
... This concept was further investigated by Hayes et al., who performed frequency and phase filtering by lock-in amplification using the rotating chains to encode the phase and frequency for the lock-in processing. The output from the lock-in amplification was determined by particle-bound fluorescent labels only, offering impressing LODs (for immunoassays of myoglobin) down to sub-femtomolar level (Mahanti et al., 2011;Petkus et al., 2006;Woolley and Hayes, 2015). ...
In comparison to alternative nanomaterials, magnetic micron/nano-sized particles show unique advantages, e.g., easy manipulation, stable signal, and high contrast. By applying magnetic actuation, magnetic particles exert forces on target objects for highly selective operation even in non-purified samples. We herein describe a subgroup of magnetic biosensors, namely optomagnetic biosensors, which employ alternating magnetic fields to generate periodic movements of magnetic labels. The optical modulation induced by the dynamics of magnetic labels is then analyzed by photodetectors, providing information of, e.g., hydrodynamic size changes of the magnetic labels. Optomagnetic sensing mechanisms can suppress the noise (by performing lock-in detection), accelerate the reaction (by magnetic force-enhanced molecular collision), and facilitate homogeneous/volumetric detection. Moreover, optomagnetic sensing can be performed using a low magnetic field (<10 mT) without sophisticated light sources or pickup coils, further enhancing its applicability for point-of-care tests. This review concentrates on optomagnetic biosensing techniques of different concepts classified by the magnetic actuation strategy, i.e., magnetic field-enhanced agglutination, rotating magnetic field-based particle rotation, and oscillating magnetic field-induced Brownian relaxation. Optomagnetic sensing principles applied with different actuation strategies are introduced as well. For each representative optomagnetic biosensor, a simple immunoassay strategy-based application is introduced (if possible) for methodological comparison. Thereafter, challenges and perspectives are discussed, including minimization of nonspecific binding, on-chip integration, and multiplex detection, all of which are key requirements in point-of-care diagnostics.
... Microfluidic technologies hold advantages over conventional platforms via lab-on-chip applications to detect multiple biotargets from a small volume of biological samples at a relatively shorter assay time (Beyazkilic et al., 2016;Bruls et al., 2009;Dittmer et al., 2010;Laksanasopin et al., 2015;Ng et al., 2018Ng et al., , 2017Piraino et al., 2016;Pollock et al., 2012;Sonawane et al., 2017;Song et al., 2012). The application of these principles to ELISA resulted in development of platforms for detection of various targets, including; cells, proteins, antibodies and lipids (Bruls et al., 2009;Cornaglia et al., 2014aCornaglia et al., , 2014bDittmer et al., 2010;Mani et al., 2016Mani et al., , 2011Tekin et al., 2013;Wang et al., 2014;Woolley and Hayes, 2015). Even though there have been substantial proof-of-concept studies conducted, there is still limited number of studies performed for validation of cardiac biomarker sensing platforms, using actual clinical samples; and a translation-ready, pump-and power-free, clinical device for monitoring biomarkers in circulation has yet to be demonstrated. ...
Microfluidic technologies offer new platforms for biosensing in various clinical and point-of-care (POC) applications. Currently, at the clinical settings, the gold standard diagnostic platforms for multiplexed sensing are multi-step, time consuming, requiring expensive and bulky instruments with a constant need of electricity which makes them unsuitable for resource-limited or POC settings. These technologies are often limited by logistics, costly assays and regular maintenance. Although there have been several attempts to miniaturize these diagnostic platforms, they stand short of batch fabrication and they are dependent on complementary components such as syringe pumps. Here, we demonstrated the development and clinical testing of a disposable, multiplexed sensing device (ToMMx), which is a portable, high-throughput and user-friendly microfluidic platform. It was built with inexpensive plastic materials and operated manually without requiring electrical power and extensive training. We validated this platform in a small cohort of 50 clinical samples from patients with cardiovascular diseases and healthy controls. The platform is rapid and gives quantifiable results with high sensitivity, as low as 5.29 pg/mL, from only a small sample volume (4 μL). ToMMx platform was compared side-by-side with commercial ELISA kits where the total assay time is reduced 15-fold, from 5 h to 20 min. This technology platform is broadly applicable to various diseases with well-known biomarkers in diagnostics and monitoring, especially with potential future impact at the POC settings.
... Magnetic beads served as an ideal platform candidate due to their high target-capturing efficiency, rapid-binding kinetics, and simple processing procedures (Woolley and Hayes 2015;Zhang et al. 2016;Atanasova, Vasileva, and Godjevargova 2017). Owing to these merits of magnetic beads, an increased sensitivity and short assay time would be achieved. ...
Human procalcitonin is an early diagnostic biomarker for sepsis and bacterial infections and can be used in distinguishing bacterial infections from viral infections. In this study, a colorimetric sensing platform for the rapid determination of procalcitonin was developed. The approach involves the capture of procalcitonin by immunomagnetic beads, and a detection antibody labeled with horseradish peroxidase to perform sandwich format, where it catalyzes the oxidation of 3,3′,5,5′–tetramethylbenzidine to produce the colorimetric signal. Under the optimal conditions, a detection limit of 0.04 ng/mL (3σ) was obtained within the calibration range 0.1–10 ng/mL. The proposed method was performed in less than 90 min and exhibited good specificity without interferences from other biomarkers including C-reactive protein and human serum albumin. Overall, the proposed method provided a new alternative strategy for procalcitonin detection due to its sensitive, rapid, specific, and simple characteristics. This method is suitable for rapid screening of various biomedical targets.
... In the future study, we plan to investigate applications in detecting patients' samples that could facilitate disease diagnosis and monitoring. MP-based immuno-aggregation strategies have been extensively used to achieve high sensitivity protein analysis [33][34][35][36][37][38][39][40] . The MPs are functionalized with recognition biomolecules to capture and detect target analytes. ...
Cell secretome analysis has gained increasing attention towards the development of effective strategies for disease treatment. Analysis of cell secretome enables the platform to monitor the status of disease progression, facilitating therapeutic outcomes. However, cell secretome analysis is very challenging due to its versatile and dynamic composition. Here, we report the development of two immuno-disaggregation bioassays using functionalized microparticles for the quantitative analysis of the cell secretome.
Methods: We evaluated the feasibility of our developed immuno-disaggregation bioassays using antibody-conjugated MPs and protein-conjugated MPs for the detection of target cell secretome protein. The vascular endothelial growth factor (VEGF)-165 protein was tested as a model cell secretome protein in the serum and serum-free conditions. The status of MP aggregates was examined with a light microscopy and AccuSizerTM 780 Optical Particle Sizer. The accuracy of our bioassays measurement was compared with standard ELISA method.
Results: The developed bioassays successfully detected target VEGF protein present in serum-free buffer and serum-containing complete cell culture medium with high sensitivity and specificity. Additionally, the immuno-disaggregation bioassays using antibody-conjugated MPs and protein-conjugated MPs have a wide detection range from 0.01 ng/mL to 100 ng/mL and 0.5 ng/mL to 100 ng/mL, respectively. The sensitivity of the bioassay using antibody-conjugated MPs was approximately one order of magnitude higher than the bioassay using protein-conjugated MPs.
Conclusion: Our promising results indicate the potential of the developed bioassays as powerful platforms for the quantitative analysis of cell secretome.
... Similar to the strategic approach demonstrated by other microfluidic blood manipulation works, this is both a demonstration of a new tool for integration in future systems and fulfills a niche for existing applications. The properties and layout of this system bear the potential to be integrated with high sensitivity rotor-based microimmunoassay after successfully realization of delivering a simplified protein solution [30]. ...
... Convenient and important test probes for this system come from current work in our laboratory, focusing on freely diffusible protein cardiac biomarkers from blood [30][31][32]. They serve as examples of proteins that are present in diagnostically important samples and as a general demonstration of the concept of sample preparation. ...
Blood is one of the most important biofluids used for clinical diagnostics. Cells and proteins in the blood can provide a rich source of information for the evaluation of human health. Efficient separation of blood components is a necessary process in order to minimize the interference of unwanted components during sensing, separation, and detection. In this paper, an insulator-based gradient dielectrophoretic device has been applied to separate red blood cells from model protein biomarkers for myocardial infarction in buffer. Within one min, red blood cells are largely depleted regardless of the minimum adherence on the channel wall. Considering the adhered red blood cells will not be transported further, a purified protein solution can be delivered for potential downstream processing or detection. Open image in new windowGraphical Abstractᅟ
... Here, however, the only assay that was performed strictly without any washing step was the competitive format type assay in buffer, while all other experiments included at least one washing step [221]. This is also true for the most recent study by the group, where following further refinement of their image analysis procedure [222], they demonstrate highly sensitive detection of three cardiac biomarkers (detection limits: myoglobin~360 aM, heart-type fatty acid binding protein (H-FABP)~67 fM, troponin I~42 fM) [223]. The biomarkers are spiked into buffer solutions and are detected by a sandwich immunoassay format performed on magnetically rotated particle clusters [223]. ...
... This is also true for the most recent study by the group, where following further refinement of their image analysis procedure [222], they demonstrate highly sensitive detection of three cardiac biomarkers (detection limits: myoglobin~360 aM, heart-type fatty acid binding protein (H-FABP)~67 fM, troponin I~42 fM) [223]. The biomarkers are spiked into buffer solutions and are detected by a sandwich immunoassay format performed on magnetically rotated particle clusters [223]. ...
... In summary, while immunoassays on magnetically rotated particle clusters can be in principle applied in a strictly homogeneous format [221], up to now the most sensitive results do involve washing steps [223], and it has yet to be demonstrated that the method can also be performed directly in unprocessed sample material with sufficient sensitivity and specificity. ...
The growing availability of biomarker panels for molecular diagnostics is leading to an increasing need for fast and sensitive biosensing technologies that are applicable to point-of-care testing. In that regard, homogeneous measurement principles are especially relevant as they usually do not require extensive sample preparation procedures, thus reducing the total analysis time and maximizing ease-of-use. In this review, we focus on homogeneous biosensors for the in vitro detection of biomarkers. Within this broad range of biosensors, we concentrate on methods that apply magnetic particle labels. The advantage of such methods lies in the added possibility to manipulate the particle labels by applied magnetic fields, which can be exploited, for example, to decrease incubation times or to enhance the signal-to-noise-ratio of the measurement signal by applying frequency-selective detection. In our review, we discriminate the corresponding methods based on the nature of the acquired measurement signal, which can either be based on magnetic or optical detection. The underlying measurement principles of the different techniques are discussed, and biosensing examples for all techniques are reported, thereby demonstrating the broad applicability of homogeneous in vitro biosensing based on magnetic particle label actuation.
Staphylococcus aureus (S. aureus) is a food-borne pathogenic organism that causes an array of infection diseases ranging from superficial skin infections to potentially fatal diseases. Thus quantitative detection of S. aureus with high selectivity and sensitivity is of great significance. In this study, staphylococcus protein A (SPA), which is specifically secreted on the surface of S. aureus, was selected as an epitope. On the basis of the specific recognition of SPA by chicken anti-protein A immunoglobulin Y (IgY), a facile colorimetric immunoassay for the detection of S. aureus was established. To our best knowledge, it was the first time to utilize chicken anti-protein A IgY as antibody pair for the quantitative detection of S. aureus. Chicken anti-protein A IgY was conjugated with magnetic particles for bacteria capture. Then horseradish peroxidase (HRP) labeled chicken anti-protein A IgY was employed as revealing partner to perform a sandwich format assay, followed by an HRP catalyzing colorimetric reaction. Optimized detection conditions produced a calibration curve for S. aureus ranging from 7.8 × 102 CFU/mL to 2.0 × 105 CFU/mL with a detection limit of 2.3 × 102 CFU/mL (3σ) in phosphate-buffered saline (PBS). The detection limits (3σ) in spiked samples were very close to that in PBS. The entire assay was accomplished in less than 60 min and no cross-reactivity with the other tested bacterial species was observed. These results demonstrate that the proposed method is effective for rapid and selective screening of S. aureus in food samples.
A highly sensitive fluorescence detection probe was developed by tailoring plastic antibodies on the external surface of aqueous soluble quantum dots (QDs). The target was Myoglobin (Myo), a cardiac biomarker that quenched the intrinsic fluorescent emission of cadmium telluride (CdTe) QDs capped with mercaptopropionic acid (CdTe-MPA-QDs). The QDs were incubated with the target protein and further modified with a molecularly-imprinted polymer (MIP) produced by radical polymerization of acrylamide and bisacrylamide. The main physical features of the materials were assessed by electron microscopy, dynamic light scattering (DLS), UV/Vis spectrophotometry and spectrofluorimetry. The plastic antibodies enabled Myo rebinding into the QDs with subsequent fluorescence quenching. This QD-probe could detect Myo concentrations from 0.304 to 571 pg/ml (50.6 fM to 95 pM), with a limit of detection of 0.045 pg/ml (7.6 fM). The proposed method was applied to the determination of Myo concentrations in synthetic human serum. The results obtained demonstrated the ability of the modified-QDs to determine Myo below the cut-off values of myocardial infarction. Overall, the nanostructured MIP-QDs reported herein displayed quick responses, good stability and sensitivity, and high selectivity for Myo, offering the potential to be explored as new emerging sensors for protein detection in human samples.
This study investigated the fabrication and proof of concept design demonstrating rapid and highly sensitive antigen capture utilizing electrospun polystyrene (PS) microfiber mat substrates paired with vacuum pump pressurization to induce bulk flow. This system takes advantage of the increased surface area of the electrospun polystyrene (ESPS) fiber mat substrates and the accelerated propagation of antigens through the detection platform by the vacuum pump to enable efficient and rapid antigen capture. Parameters for microfiber mat fabrication were optimized for the degree of antibody immobilization, and bulk flow was optimized for the antigen capture efficiency. The amount of antigen captured upon a traditional, flat PS substrate in 60 minutes surpassed in under 5 seconds when utilizing the microfiber-vacuum system. The results from this study demonstrate the synergistic potential of ESPS fiber mat substrates under bulk flow to enable semi-instantaneous detection of disease markers for immunoassay diagnostics.
