Flu is caused by the influenza virus that, due to mutations, keeps our body vulnerable for infections, making early diagnosis essential. Although immuno-based diagnostic tests are available, they have low sensitivity and reproducibility. In this paper, the prospect of detecting influenza A virus using digital ELISA has been studied. To appropriately select bioreceptors for this bioassay, seven commercial antibodies against influenza A nucleoprotein were methodically tested for their reactivity and binding affinity. The study has been performed on two markedly different platforms, being an enzyme-linked immunosorbent assay and a surface plasmon resonance system. The selected antibodies displayed completely different behavior on the two platforms and in various assay configurations. Surprisingly, the antibodies that showed overall good reactivity on both platforms had the highest dissociation constant among the tested antibodies, suggesting that, although important, binding affinity is not the only parameter to be considered when selecting antibodies. Moreover, only one antibody had the capacity to capture the nucleoprotein directly in lysis buffer used for releasing this viral protein, which might pose a huge advantage when developing assays with a fast time-to-result. This antibody was implemented on an in-house developed digital ELISA platform for ultrasensitive detection of recombinant nucleoprotein, reaching a detection limit of 4 ± 1 fM in buffer and 10 ± 2 fM in 10-fold diluted nasopharyngeal swabs, which is comparable to currently available fast molecular detection techniques. These results point to a great potential for ultrasensitive immuno-based influenza detection.
The close correlation between Tau pathology and Alzheimer's disease (AD) progression makes this protein a suitable biomarker for diagnosis and monitoring of the disorder evolution. However, the use of Tau in diagnostics has been hampered, as it currently requires collection of cerebrospinal fluid (CSF), which is an invasive clinical procedure. Although measuring Tau-levels in blood plasma would be favorable, the concentrations are below the detection limit of a conventional ELISA. In this work, we developed a digital ELISA for the quantification of attomolar protein Tau concentrations in both buffer and biological samples. Individual Tau molecules were first captured on the surface of magnetic particles using in-house developed antibodies and subsequently isolated into the femtoliter-sized wells of a 2 × 2 mm microwell array. Combination of high-affinity antibodies, optimal assay conditions and a digital quantification approach resulted in a 24 ± 7 aM limit of detection (LOD) in buffer samples. Additionally, a dynamic range of 6 orders of magnitude was achieved by combining the digital readout with an analogue approach, allowing quantification from attomolar to picomolar levels of Tau using the same platform. This proves the compatibility of the presented assay with the wide range of Tau concentrations encountered in different biological samples. Next, the developed digital assay was applied to detect total Tau levels in spiked blood plasma. A similar LOD (55 ± 29 aM) was obtained compared to the buffer samples, which was 5000-fold more sensitive than commercially available ELISAs and even outperformed previously reported digital assays with 10-fold increase in sensitivity. Finally, the performance of the developed digital ELISA was assessed by quantifying protein Tau in three clinical CSF samples. Here, a high correlation (i.e. Pearson coefficient of 0.99) was found between the measured percentage of active particles and the reference protein Tau values. The presented digital ELISA technology has great capacity in unlocking the potential of Tau as biomarker for early AD diagnosis.
The lab-on-a-chip (LOC) field has witnessed an excess of new technology concepts, especially for the point-of-care (POC) applications. However, only few concepts reached the POC market often because of challenging integration with pumping and detection systems as well as with complex biological assays. Recently, a new technology termed SIMPLE was introduced as a promising POC platform due to its features of being self-powered, autonomous in liquid manipulations, cost-effective and amenable to mass production. In this paper, we improved the SIMPLE design and fabrication and demonstrated for the first time that the SIMPLE platform can be successfully integrated with biological assays by quantifying creatinine, biomarker for chronic kidney disease, in plasma samples. To validate the robustness of the SIMPLE technology, we integrated a SIMPLE-based microfluidic cartridge with colorimetric read-out system into the benchtop Creasensor. This allowed us to perform on-field validation of the Creasensor in a single-blind study with 16 plasma samples, showing excellent agreement between measured and spiked creatinine concentrations (ICC: 0.97). Moreover, the range of clinically relevant concentrations (0.76-20 mg/dL), the sample volume (5 μL) and time-to-result of only 5 min matched the Creasensor performance with both lab based and POC benchmark technologies. This study demonstrated for the first time outstanding robustness of the SIMPLE in supporting the implementation of biological assays. The SIMPLE flexibility in liquid manipulation and compatibility with different sample matrices opens up numerous opportunities for implementing more complex assays and expanding its POC applications portfolio.
Aptamers are emerging as powerful synthetic bioreceptors for fundamental research, diagnostics, and therapeutics. For further advances, it is important to gain a better understanding of how aptamers interact with their targets. In this work, we have used magnetic force-induced dissociation experiments to study the dissociation process of two different aptamer−protein complexes, namely for hIgE and Ara h 1. The measurements show that both complexes exhibit dissociation with two distinct regimes: the dissociation rate depends weakly on the applied force at high forces but depends stronger on force at low forces. We attribute these observations to the existence of at least one intermediate state and at least two energy barriers in the aptamer−protein interaction. The measured spontaneous dissociation rate constants were validated with SPR using both Biacore and fiber optic technology. This work demonstrates the potential of the magnetic forceinduced dissociation approach for an in-depth study of the dissociation kinetics of aptamer−protein bonds, which is not possible with SPR technologies. The results will help in the development and expansion of aptamers as bioaffinity probes.
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