Highly Sensitive Multiplex Detection of Molecular Biomarkers Using Hybridization Chain Reaction in an Encoded Particle Microfluidic Platform.

In the continuous combat against diseases, there is the need for tools that enable an improved diagnostic efficiency towards higher information density combined with reduced time-to-result and cost. Here, a novel fully integrated microfluidic platform, the Evalution™, is evaluated as a potential solution to this need. Encoded microparticles combined with channel-based microfluidics allow a fast, sensitive and simultaneous detection of several disease-related biomarkers. Since the binary code is represented by physically present holes, 210 different codes can be created that will not be altered by light or chemically induced degradation. Exploiting the unique features of this multiplex platform, hybridization chain reaction (HCR) is explored as a generic approach to reach the desired sensitivity. Compared to a non-amplified reference system, the sensitivity was drastically improved by a factor of 104, down to low fM LOD values. Depending on the HCR duration, the assay can be tuned for sensitivity or total assay time, as desired. The huge potential of this strategy was further demonstrated by the successful detection of a multiplex panel of six different nucleic acid targets including viruses and bacteria. The ability to not only discriminate these two categories but, with the same effort, also virus strains (human adenovirus and human bocavirus), virus subtypes (human adenovirus type B and D) and antibiotic-resistant bacteria (Streptococcus pneumonia), exemplifies the specificity of the developed approach. The effective, yet highly simplified, isothermal and protein-enzyme-free signal amplification tool reaches an LOD ranging from as low as 33 ± 4 to 151 ± 12 fM for the different targets. Moreover, direct detection in a clinically relevant sample matrix was verified, resulting in a detection limit of 309 ± 80 fM, approximating the low fM levels detectable with the gold standard analysis method, PCR, without the drawbacks related to protein enzymes, thermal cycling and elaborate sample preparation steps. The reported strategy can be directly transferred as a generic approach for the sensitive and specific detection of various target molecules in multiplex. In combination with the high-throughput capacity and reduced reagent consumption, the Evalution™ demonstrates immense potential in the next generation of diagnostic tools towards more personalized medicine.

Bridging the Gap between Digital Assays and Point-of-Care Testing: Automated, Low Cost, and Ultrasensitive Detection of Thyroid Stimulating Hormone.

Medical diagnostics is moving toward disease-related target detection at very low concentrations because of the (1) quest for early-stage diagnosis, at a point where only limited target amounts are present, (2) trend toward minimally invasive sample extraction, yielding samples containing low concentrations of target, and (3) need for straightforward sample collection, usually resulting in limited volume collected. Hence, diagnostic tools allowing ultrasensitive target detection at the point-of-care (POC) are crucial for simplified and timely diagnosis of many illnesses. Therefore, we developed an innovative, fully integrated, semi-automated, and economically viable platform based on (1) digital microfluidics (DMF), enabling automated manipulation and analysis of very low sample volumes and (2) low-cost disposable DMF chips with microwell arrays, fabricated via roll-to-roll processes and allowing digital target counting. Thyroid stimulating hormone detection was chosen as a relevant application to show the potential of the system. The assay buffer was selected using design of experiments, and the assay was optimized in terms of reagent concentration and incubation time toward maximum sensitivity. The hydrophobic-in-hydrophobic microwells showed an unparalleled seeding efficiency of 97.6% ± 0.6%. A calculated LOD of 0.0013 µIU/mL was obtained, showing the great potential of the platform, especially taking into account the very low sample volume analyzed (1.1 µL). Although validation (in biological matrix) and industrialization (full automation) steps still need to be taken, it is clear that the combination of DMF, low-cost DMF chips, and digital analyte counting in microwell arrays enables the implementation of ultrasensitive and reliable target detection at the POC.

Engineered tracrRNA for enabling versatile CRISPR-dCas9-based biosensing concepts.

In recent years, CRISPR-Cas (stands for: clustered regularly interspaced short palindromic repeats - CRISPR associated protein) based technologies have gained increasing attention in the biosensing field. Thanks to excellent sequence specificity, their use is of particular interest for detecting nucleic acid (NA) targets. In this context, signal generation and amplification can be realized by employing the cis-cleavage activity of the Cas9 protein, although other options involving the catalytically inactive dead Cas9 (dCas9) are increasingly explored. The latter are however mostly based on complex protein engineering processes and often lack efficient signal amplification. Here we showed for the first time that flexible signal generation and amplification properties can be integrated into the CRISPR-dCas9 complex based on a straightforward incorporation of a DNA sequence into the trans-activating CRISPR RNA (tracrRNA). The intrinsic nuclease activity of the engineered complex remained conserved, while the incorporated DNA stretch enabled two modes of amplified fluorescent signal generation: (1) as an RNA-cleaving DNA-based enzyme (DNAzyme) or (2) as hybridization site for biotinylated DNA probes, allowing subsequent enzyme labeling. Both signal generation strategies were demonstrated in solution as well as while coupled to a solid surface. Finally, in a proof of concept bioassay, we demonstrated the successful detection of single stranded DNA on magnetic microbeads using the engineered CRISPR-dCas9 complex. Thanks to the flexibility of incorporating different NA-based signal generation and amplification strategies, this novel NA engineering approach holds enormous promise for many new CRISPR-based biosensing applications.

Innovative FO-SPR Label-free Strategy for Detecting Anti-RBD Antibodies in COVID-19 Patient Serum and Whole Blood.

The ongoing COVID-19 pandemic has emphasized the urgent need for rapid, accurate, and large-scale diagnostic tools. Next to this, the significance of serological tests (i.e., detection of SARS-CoV-2 antibodies) also became apparent for studying patients' immune status and past viral infection. In this work, we present a novel approach for not only measuring antibody levels but also profiling of binding kinetics of the complete polyclonal antibody response against the receptor binding domain (RBD) of SARS-CoV-2 spike protein, an aspect not possible to achieve with traditional serological tests. This fiber optic surface plasmon resonance (FO-SPR)-based label-free method was successfully accomplished in COVID-19 patient serum and, for the first time, directly in undiluted whole blood, omitting the need for any sample preparation. Notably, this bioassay (1) was on par with FO-SPR sandwich bioassays (traditionally regarded as more sensitive) in distinguishing COVID-19 from control samples, irrespective of the type of sample matrix, and (2) had a significantly shorter time-to-result of only 30 min compared to >1 or 4 h for the FO-SPR sandwich bioassay and the conventional ELISA, respectively. Finally, the label-free approach revealed that no direct correlation was present between antibody levels and their kinetic profiling in different COVID-19 patients, as another evidence to support previous hypothesis that antibody-binding kinetics against the antigen in patient blood might play a role in the COVID-19 severity. Taking all this into account, the presented work positions the FO-SPR technology at the forefront of other COVID-19 serological tests, with a huge potential toward other applications in need for quantification and kinetic profiling of antibodies.

Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry.

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

Encoded particle microfluidic platform for rapid multiplexed screening and characterization of aptamers against influenza A nucleoprotein.

Aptamers represent interesting bioreceptor alternatives to antibodies when developing a bioassay and are selected by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process. After selection, an extensive characterization process is essential to verify the binding affinity and specificity of aptamer candidates, which is the most time-consuming and costly step. In this study, we assessed a new microfluidic platform, namely Evalution™, as a rapid and high throughput aptamer characterization platform. To do this, we first selected aptamers against influenza A nucleoprotein (infA NP) by performing magnetic bead-based SELEX. The selected aptamer candidates were subsequently screened using Evalution™ for their binding kinetics and specificity towards infA NP. All aptamers showed dissociation constants (KD) in the low nanomolar range (from 13 to 41 nM), and differential binding behavior towards control proteins, such as BSA and influenza B nucleoprotein (infB NP). Among 5 selected candidates, one aptamer (NP5) exhibited a significant discrimination between infA NP and infB NP and was further used to benchmark the kinetic analysis of Evalution™ (KD = 41 nM) with an SPR platform (KD = 17 nM). These results suggested that NP5 has the potential to be used for developing sensitive and infA NP specific aptamer-based assay. Moreover, the presented platform proved to be an efficient aptamer characterization tool for performing typical aptamer characterization experiments like binding kinetics (due to the real-time monitoring feature) and specificity assessment in a high-throughput manner due to the multiplexing capacity.

Digital ELISA for the quantification of attomolar concentrations of Alzheimer's disease biomarker protein Tau in biological samples.

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 mm2 microwell array. Combination of high-affinity antibodies, optimal assay conditions and a digital quantification approach resulted in a 24 ±â€¯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 ±â€¯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.

Target Confinement in Small Reaction Volumes Using Microfluidic Technologies: A Smart Approach for Single-Entity Detection and Analysis.

Over the last decades, the study of cells, nucleic acid molecules, and proteins has evolved from ensemble measurements to so-called single-entity studies. The latter offers huge benefits, not only as biological research tools to examine heterogeneities among individual entities within a population, but also as biosensing tools for medical diagnostics, which can reach the ultimate sensitivity by detecting single targets. Whereas various techniques for single-entity detection have been reported, this review focuses on microfluidic systems that physically confine single targets in small reaction volumes. We categorize these techniques as droplet-, microchamber-, and nanostructure-based and provide an overview of their implementation for studying single cells, nucleic acids, and proteins. We furthermore reflect on the advantages and limitations of these techniques and highlight future opportunities in the field.

Fast multiplex analysis of antibodies in complex sample matrix using the microfluidic Evalution™ platform.

Biosensor development requires comprehensive research for establishing the optimal bioassay conditions that determine the final kinetics, sensitivity and specificity. Different systems have been developed to study bioreceptor-target interactions but they often have drawbacks, such as long hands-on time, low throughput, high sample consumption and high cost. In this work, the potential of the novel microfluidic Evalution™ platform has been evaluated for developing sandwich-based assays in a fast and high-throughput fashion. An immunoassay for the detection of influenza A nucleoprotein was used as a model system. Exploiting the platform's unique features, various typical bioassay parameters (e.g. aspecific binding between assay components, different reagent concentrations and incubation times) were tested for three capture antibodies in a simple and fast manner (2 runs of 80 min). The selected conditions, giving the highest signal-to-noise ratio, were directly employed on the same platform to detect nucleoprotein in buffer and nasopharyngeal swabs. Two antibodies with a higher dissociation constant (Ab11 and Ab12) required longer incubation times (60 min) for sensitive detection (limit of detection (LOD) of 0.48 and 0.26 ng mL-1, respectively) compared to an antibody with lower dissociation constant (LOD of 0.04 ng mL-1 for Ab66 within 30 min). Moreover, one antibody (Ab12) showed limited capacity to capture nucleoprotein directly in sample matrix. The obtained results were in accordance with previous studies performed on an ELISA and SPR platform with the same antibodies. This positions the Evalution™ platform as a reliable platform for fast and multiplex analysis of antibodies' performance both in buffer and complex sample matrices.

Bioassay Development for Ultrasensitive Detection of Influenza A Nucleoprotein Using Digital ELISA.

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.

reMelting curve analysis as a tool for enrichment monitoring in the SELEX process.

Current aptamer selection procedures enable limited control and transparency on how the DNA selection pool is evolving. Affinity tests and binding analyses are not always informative. Here we show that real-time PCR provides a valuable tool for the follow-up of aptamer selection. Limited time, work and amount of amplified ssDNA make this an interesting instrument to set-up a SELEX design and monitor the enrichment of oligonucleotides. reMelting Curve Analysis (rMCA) after reannealing under stringent conditions provides information about enrichment, compared to a random library. Monitoring the SELEX process and optimising conditions by means of the proposed methods can increase the selection efficiency in a controlled way. rMCA is applied in enrichment simulations and three different selection procedures. Our results imply that rMCA can be used for different SELEX designs and different targets. SELEX pool diversity analysis by rMCA has been proven to be a useful, reproducible tool to detect and evaluate enrichment of specific binding aptamers while the selection procedure is being performed.