Integrating Magnetic-Bead-Based Sample Extraction and Molecular Barcoding for the One-Step Pooled RT-qPCR Assay of Viral Pathogens without Retesting.

Pooling multiple samples prior to real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis has been proposed as a strategy to minimize expenses and boost test throughput during the COVID-19 pandemic. Nevertheless, the traditional pooling approach cannot be effectively deployed in high-prevalence settings due to the need for secondary tests in the case of a positive pool. In this study, we present a pooling test platform with high adaptability and simplicity that allows sample-specific detection of multiple-tagged samples in a single run without the need for retesting. This was accomplished by labeling distinct samples with predefined ID-Primers and identifying tagged pooled samples using one-step RT-PCR followed by melting curve analysis with rationally designed universal fluorescence- and quencher-tagged oligo probes. Using magnetic beads (MBs), nucleic acid targets from different individuals can be tagged and extracted concurrently and then pooled before RT, eliminating the need for extra RNA extraction and separate RT and enzyme digestion steps in the recently developed barcoding strategies. Pools of six samples (positive and negative) were successfully identified by melting temperature values under two fluorescent channels, with a detection sensitivity of 5 copies/µL. We validated the reproducibility of this assay by running it on 40 clinical samples with a hypothetical infection rate of 15%. In addition, to aid the scenario of large-scale pooling tests, we constructed a melting curve autoreadout system (MCARS) for statistical analysis of melting curve plots to eliminate error-prone manual result readout. Our results suggest that this strategy could be a simple and adaptable tool for alleviating existing bottlenecks in diagnostic pooling testing.

Efficient large-scale screening of viral pathogens by fragment length identification of pooled nucleic acid samples (FLIPNAS).

The necessity for the large-scale screening of viral pathogens has been amply demonstrated during the COVID-19 pandemic. During this time, SARS-CoV-2 nucleic acid pooled testing, such as Dorfman-based group testing, was widely adopted in response to the sudden increased demand for detection. However, the current approach still necessitates the individual retesting of positive pools. Here, we established an efficient method termed the fragment-length identification of pooled nucleic acid samples (FLIPNAS), where all subsamples (n = 8) can be uniquely labelled and tested in a single-time detection among pools of samples. We used a novel and simple design of unique primers (UPs) to generate amplicons of unique lengths after reverse transcription and polymerase chain reaction to reach this aim. As a result, the unique lengths of the amplicons can be recognized and traced back to the corresponding UPs and specific samples. Our results demonstrated that FLIPNAS could recognize one to eight positive subsamples in a single test without retesting positive pools. The system also showed sufficient sensitivity for the mass monitoring of SARS-CoV-2 and no cross-reactivity against three common respiratory diseases. Moreover, the FLIPNAS results of 40 samples with a positive ratio of 7.8% were in 100% agreement with their individual detection results using the gold standard. Collectively, this study shows that the efficiency of nucleic acid pooling detection can be further improved by FLIPNAS, which can speed up testing and mitigate the urgent demand for resources.

Unique Barcoded Primer-Assisted Sample-Specific Pooled Testing (Uni-Pool) for Large-Scale Screening of Viral Pathogens.

Pooled testing has been widely adopted recently to facilitate large-scale community testing during the COVID-19 pandemic. This strategy allows to collect and screen multiple specimen samples in a single test, thus immensely saving the assay time and consumable expenses. Nevertheless, when the outcome of a pooled testing is positive, it necessitates repetitive retesting steps for each sample which can pose a serious challenge during a rising infection wave of increasing prevalence. In this work, we develop a unique barcoded primer-assisted sample-specific pooled testing strategy (Uni-Pool) where the key genetic sequences of the viral pathogen in a crude sample are extracted and amplified with concurrent tagging of sample-specific identifiers. This new process improves the existing pooled testing by eliminating the need for retesting and allowing the test results-positive or negative-for all samples in the pool to be revealed by multiplex melting curve analysis right after real-time polymerase chain reaction. It significantly reduces the total assay time for large-scale screening without compromising the specificity and detection sensitivity caused by the sample dilution of pooling. Our method was able to successfully differentiate five samples, positive and negative, in one pool with negligible cross-reactivity among the positive and negative samples. A pooling of 40 simulated samples containing severe acute respiratory syndrome coronavirus-2 pseudovirus of different loads (min: 10 copies/µL; max: 103 copies/µL) spiked into artificial saliva was demonstrated in eight randomized pools. The outcome of five samples in one pool with a hypothetical infection prevalence of 15% in 40 samples was successfully tested and validated by a typical Dorman-based pooling.

Prediction of Aptamer-Small-Molecule Interactions Using Metastable States from Multiple Independent Molecular Dynamics Simulations.

Understanding aptamer-ligand interactions is necessary to rationally design aptamer-based systems. Commonly used in silico tools have proven to be accurate to predict RNA and DNA oligonucleotide tertiary structures. However, given the complexity of nucleic acids, the most thermodynamically stable conformation is not necessarily the one with the highest affinity for a specific ligand. Because many metastable states may coexist, it remains challenging to predict binding sites through molecular docking simulations using available computational pipelines. In this study, we used independent simulations to broaden the conformational diversity sampled from DNA initial models of distinct stability and assessed the binding affinity of selected metastable representative structures. In our results, utilizing multiple metastable conformations for molecular docking analysis helped identify structures favorable for ligand binding and accurately predict the binding sites. Our workflow was able to correctly identify the binding sites of the characterized adenosine monophosphate and l-argininamide aptamers. Additionally, we demonstrated that our pipeline can be used to aid the design of competition assays that are conducive to aptasensing strategies using an uncharacterized aflatoxin B1 aptamer. We foresee that this approach may help rationally design effective and truncated aptamer sequences interacting with protein biomarkers or small molecules of interest for drug design and sensor applications.

An Insight into Tunable Innate Piezoelectricity of Silk for Green Bioelectronics.

Commercially available bioelectronics account for significant percentage of e-waste, especially battery waste, that demand immediate intervention due to rising environmental concerns. Consumers are becoming increasingly aware and cautious of their contribution to carbon footprint on a regular basis. It has become imperative to adopt sustainability in every aspect of production of bioelectronics taking into consideration the growing market for wearable healthcare monitoring system. Green electronics is a relatively new concept gaining tremendous attention within the scientific and industrial community with the ultimate goal of employing organic, biodegradable, and self-sustainable system to replace the conventional inorganic battery-powered electronics. Silk is a green material that has been extensively explored for its use in functional electronics due to its tunable biodegradability and flexibility. Nevertheless, an intriguing property of Silk is its innate piezoelectricity. This review highlights the importance of crystal orientation and structure of Silk Fibroin to display piezoelectric response and documents possible strategies for its enhancement. It also provides insight into the possibility of using piezoelectric Silk as a piezoelectric sensor, actuator, and energy harvester to form self-powered hybrid systems for autonomous bioelectronics.

Rational design of allosterically regulated toehold mediated strand displacement circuits for sensitive and on-site detection of small molecule metabolites.

Development of small molecule biosensors enables rapid and de-centralized small molecule detection that meets the demands of routine health monitoring and rapid diagnosis. Among them, allosteric transcription factor (aTF)-based biosensors have shown potential in modular design of small molecule detection platforms due to their ligand-regulated DNA binding activity. Here, we expand the capabilities of a biosensor that leverages the aTF-based regulation of toehold-mediated strand displacement (TMSD) circuits for uric acid (UA) detection in non-invasive salivary samples by utilizing the UA-responsive aTF HucR. The impact of the low ligand affinity of the native HucR was addressed by engineering a two-pass TMSD circuit with in silico rational design. This combined strategy achieved enrichment of the output signal and overcame the negative impact of the matrix effect on the sensitivity and overall response of the biosensor when using real samples, which enabled semi-quantitative detection in the normal salivary UA levels. As well, enhancements provided by the two-pass design halved the turnaround time to less than 15 minutes. To sum up, the two-cycle DNA circuit design enabled aTF-based simple, rapid and one-step non-invasive salivary UA detection, showing its potential in metabolite detection for health monitoring.

B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release.

Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Nucleic Acid Self-Assembly Circuitry Aided by Exonuclease III for Discrimination of Single Nucleotide Variants.

Robust and rapid discrimination of one base mutations in nucleic acid sequences is important in clinical applications. Here, we report a hybridization-based assay exploiting nucleic acid self-assembly circuitry and enzyme exonuclease III (Exo III) for the differentiation of single nucleotide variants (SNVs). This one-step approach combines the merits of discrimination power of competitive DNA hybridization probes (probe + sink) with catalytic amplification assisted by Exo III. The phosphorothioate bonds modified on a wild-type (WT) specific sink inhibit the Exo III digestion; thus, subsequent catalytic amplification magnifies only the intended SNV targets. The integrated assay exhibits improved SNV discrimination rather than hybridization probes relying solely on competition or amplification and enables SNV detection at 1% abundance. Two frequent cancer-driver mutation sequences (EGFR-L861Q, NRAS-Q61K) were tested. Our strategy allows simple sequence design and can easily adapt to multianalyte SNV detections.

Conditional Displacement Hybridization Assay for Multiple SNP Phasing.

The two chromosomal copies of the human genome are highly polymorphic, and the allelic content on each strand can dictate a person's biological outcomes. While many of the current diagnostic tools are able to detect the presence of multiple mutations at the same time, most cannot determine the phase of these mutations unless long-range PCR or sequencing techniques are used or if templates are compartmentalized into single copies prior to amplification. Here, an enzyme-coupled hybridization assay, named conditional displacement hybridization assay (CDHA), is described for the concurrent and rapid determination of the presence and phase of SNP variants. In this approach, short DNA probes were utilized to first quantify the amount of SNPs on the templates using a two-channel fluorescence measurement. The hybrids formed between the probes and the templates then set up the right condition for the subsequent enzymatic displacement and quenching of a fluorophore-labeled strand, which happens only if both SNPs are present on the same strand. The drop in the fluorescence signal thereby indicates the phase of the two SNPs. As a proof of concept, we tested the assay on four variants of an arbitrary sequence-with or without mutation on two sites 100 nts apart. The assay described herein was able to determine the haplotype phase of the samples in less than 1 h. This method promises a direct, cost-effective, and laboratory-based test to extract further genetic information to determine and/or predict diseases and traits dependent on SNP phasing.

Kinetically modulated specificity against single-base mutants in nucleic acid recycling circuitry using the destabilization motif.

Signal amplification in nucleic acid sensing improves detection sensitivity, but difficulties remain in sustaining specificity over time, particularly under excess amounts of single-base mutants. Here, we report simple, self-refining target recycling circuitry, which cumulates differentiation between on and off targets by 2-step cyclic interaction with the sensing probe. In the reaction, the analyte recycles only if the protective strand of the sensing probe is removed. The dissociation kinetics of such interaction was modulated by reacting it with different lengths of assistant strands. When shorter assistant strands are used, the destabilization motif of the sensing probe has to spontaneously dissociate before another assistant strand approaches and fully displaces it. This sets up a high kinetic barrier sensitive to the subtle reaction energy differences imposed by the single-base mutants, and substantially improved specificity. As a proof of concept, a microRNA 21 DNA analogue was chosen as our target analyte together with its 14 point mutants (substitution, insertion, or deletion) for specificity measurements. The experimental results corroborate that our system amplifies signals in a comparable manner to the traditional one-layer recycling approach but with negligible system leakage. With the use of shortened assistant strands, up to 100 fold increase in the discrimination factor against the single-base mutants is observed. Specificity is sustainable or even increased over long period measurements (i.e. 4 days). More importantly, target differentiation is successfully demonstrated even in excess amounts of spurious analogs (100×) and low target frequency mixtures (i.e. 0.1%), which mimic the lean conditions practically encountered. Explicit mechanisms of the system specificity are elucidated through analytical calculations and free energy level diagrams. The modularity of the destabilization motif herein promises detection of different nucleic acid based targets and integration into other signal amplification approaches for specificity enhancement.

Kinetically-enhanced DNA detection via multiple-pass exonuclease III-aided target recycling.

One of the promising approaches to address the challenge of detecting dilute nucleic acid analytes is exonuclease III-aided target recycling. In this strategy, the target DNA self-assembles with the reactant DNA probes and displays itself as a reactant and product at the same time. This provides an autonomous mechanism to release and reuse the analyte from each round of reactions for repetitive cycles, which amplifies the signal without amplifying the analyte itself. However, for very low amounts of the analyte, it takes a considerably long time before a detectable signal is generated. Thus, in this paper, we report a kinetically-enhanced target recycling strategy by designing two more target recycling sub-reactions that are triggered by the byproducts of the first reaction involving the target analyte. In this manner, concentrations of up to 0.5 pM of target DNA can be detected in 15 minutes.

Statistical linkage analysis of substitutions in patient-derived sequences of genotype 1a hepatitis C virus nonstructural protein 3 exposes targets for immunogen design.

UNLABELLED: Chronic hepatitis C virus (HCV) infection is one of the leading causes of liver failure and liver cancer, affecting around 3% of the world's population. The extreme sequence variability of the virus resulting from error-prone replication has thwarted the discovery of a universal prophylactic vaccine. It is known that vigorous and multispecific cellular immune responses, involving both helper CD4(+) and cytotoxic CD8(+) T cells, are associated with the spontaneous clearance of acute HCV infection. Escape mutations in viral epitopes can, however, abrogate protective T-cell responses, leading to viral persistence and associated pathologies. Despite the propensity of the virus to mutate, there might still exist substitutions that incur a fitness cost. In this paper, we identify groups of coevolving residues within HCV nonstructural protein 3 (NS3) by analyzing diverse sequences of this protein using ideas from random matrix theory and associated methods. Our analyses indicate that one of these groups comprises a large percentage of residues for which HCV appears to resist multiple simultaneous substitutions. Targeting multiple residues in this group through vaccine-induced immune responses should either lead to viral recognition or elicit escape substitutions that compromise viral fitness. Our predictions are supported by published clinical data, which suggested that immune genotypes associated with spontaneous clearance of HCV preferentially recognized and targeted this vulnerable group of residues. Moreover, mapping the sites of this group onto the available protein structure provided insight into its functional significance. An epitope-based immunogen is proposed as an alternative to the NS3 epitopes in the peptide-based vaccine IC41. IMPORTANCE: Despite much experimental work on HCV, a thorough statistical study of the HCV sequences for the purpose of immunogen design was missing in the literature. Such a study is vital to identify epistatic couplings among residues that can provide useful insights for designing a potent vaccine. In this work, ideas from random matrix theory were applied to characterize the statistics of substitutions within the diverse publicly available sequences of the genotype 1a HCV NS3 protein, leading to a group of sites for which HCV appears to resist simultaneous substitutions possibly due to deleterious effect on viral fitness. Our analysis leads to completely novel immunogen designs for HCV. In addition, the NS3 epitopes used in the recently proposed peptide-based vaccine IC41 were analyzed in the context of our framework. Our analysis predicts that alternative NS3 epitopes may be worth exploring as they might be more efficacious.

Triggering hairpin-free chain-branching growth of fluorescent DNA dendrimers for nonlinear hybridization chain reaction.

We present a nonlinear hybridization chain reaction (HCR) system in which a trigger DNA initiates self-sustained assembly of quenched double-stranded substrates into fluorescent dendritic nanostructures. During the process, an increasing number of originally sequestered trigger sequences labeled with fluorescent reporters are freed up from quenched substrates, leading to chain-branching growth of the assembled DNA dendrimers and an exponential increase in the fluorescence intensity. The triggered assembly behavior was examined by PAGE analysis, and the morphologies of the grown dendrimers were verified by AFM imaging. The exponential kinetics of the fluorescence accumulation was also confirmed by time-dependent fluorescence spectroscopy. This method adopts a simple sequence design strategy, the concept of which could be adapted to program assembly systems with higher-order growth kinetics.

Benchtop to at-home test: Amplicon-depleted CRISPR-regulated loop mediated amplification at skin-temperature for viral load monitoring.

CoRPLA (CRISPR-regulated One-pot Recombinase Polymerase Loop-mediated Amplification) is an amplicon-depleted skin-temperature operated iNAAT designed for at-home testing. It uses specially designed loop primers to enhance isothermal amplification, triggering Cas12 for in-situ amplicon depletion and signal amplification. This method addresses issues like amplicon-derived aerosol contamination and complex assay formats, enabling quantitative detection with sub-attomolar sensitivity (0.5 cps/µL). CoRPLA employs a DNA hydrogel wearable tape for real-time, colorimetric readout, allowing visual differentiation of pathogen loads. It was validated with clinical samples for SARS-CoV-2, RSV, influenza A, and HPV, successfully identifying multi-level viral loads of the positive cases with results consistent with qPCR. Offering high sensitivity while eliminating false positives from aerosol contamination, CoRPLA bridges the molecular assay from benchtop to home for daily viral infections monitoring.

Conformation-dependent exonuclease III activity mediated by metal ions reshuffling on thymine-rich DNA duplexes for an ultrasensitive electrochemical method for Hg2+ detection.

Hg(2+) is known to bind very strongly with T-T mismatches in DNA duplexes to form T-Hg(2+)-T base pairs, the structure of which is stabilized by covalent N-Hg bonds and exhibits bonding strength higher than hydrogen bonds. In this work, we exploit exonuclease III (Exo III) activity on DNA hybrids containing T-Hg(2+)-T base pairs and our experiments show that Hg(2+) ions could intentionally trigger the activity of Exo III toward a designed thymine-rich DNA oligonucleotide (e-T-rich probe) by the conformational change of the probe. Our sensing strategy utilizes this conformation-dependent activity of Exo III, which is controlled through the cyclical shuffling of Hg(2+) ions between the solution phase and the solid DNA hybrid. This interesting attribute has led to the development of an ultrasensitive detection platform for Hg(2+) ions with a detection limit of 0.2 nM and a total assay time within minutes. This simple detection strategy could be used for the detection of other metal ions which exhibit specific interactions with natural or synthetic bases.

CoLAMP: CRISPR-based one-pot loop-mediated isothermal amplification enables at-home diagnosis of SARS-CoV-2 RNA with nearly eliminated contamination utilizing amplicons depletion strategy.

Rapid point-of-care diagnostics, essential in settings such as airport on-site testing and home-based screening, displayed important implications for infectious disease control during the SARS-CoV-2 outbreak. However, the deployment of simple and sensitive assays in real-life scenarios still faces the concern of aerosol contamination. Here, we report an amplicon-depleting CRISPR-based one-pot loop-mediated isothermal amplification (CoLAMP) assay for point-of-care diagnosis of SARS-CoV-2 RNA. In this work, AapCas12b sgRNA is designed to recognize the activator sequence sited in the loop region of the LAMP product, which is crucial for exponential amplification. By destroying the aerosol-prone amplifiable products at the end of each amplification reaction, our design can significantly reduce the amplicons contamination that causes false positive results in point-of-care diagnostics. For at-home self-testing, we designed a low-cost sample-to-result device for fluorescence-based visual interpretation. As well, a commercial portable electrochemical platform was deployed as a proof-of-concept of ready-to-use point-of-care diagnostic systems. The field deployable CoLAMP assay can detect as low as 0.5 copies/µL of SARS-CoV-2 RNA in clinical nasopharyngeal swab samples within 40 min without the need for specialists for its operation.

Ultrasensitive solution-phase electrochemical molecular beacon-based DNA detection with signal amplification by exonuclease III-assisted target recycling.

Taking advantage of the preferential exodeoxyribonuclease activity of exonuclease III in combination with the difference in diffusivity between an oligonucleotide and a mononucleotide toward a negatively charged ITO electrode, a highly sensitive and selective electrochemical molecular beacon (eMB)-based DNA sensor has been developed. This sensor realizes electrochemical detection of DNA in a homogeneous solution, with sensing signals amplified by an exonuclease III-based target recycling strategy. A hairpin-shaped oligonucleotide containing the target DNA recognition sequence, with a methylene blue tag close to the 3' terminus, is designed as the signaling probe. Hybridization with the target DNA transforms the probe's exonuclease III-inactive protruding 3' terminus into an exonuclease III-active blunt end, triggering the digestion of the probe into mononucleotides including a methylene blue-labeled electro-active mononucleotide (eNT). The released eNT, due to its less negative charge and small size, diffuses easily to the negative ITO electrode, resulting in an increased electrochemical signal. Meanwhile, the intact target DNA returns freely to the solution and hybridizes with other probes, releasing multiple eNTs and thereby further amplifies the electrochemical signal. This new immobilization-free, signal-amplified electrochemical DNA detection strategy shows great potential to be integrated in portable and cost-effective DNA sensing devices.

Staining-free gel electrophoresis-based multiplex enzyme assay using DNA and peptide dual-functionalized gold nanoparticles.

We report a simple staining-free gel electrophoresis method to simultaneously probe protease and nuclease. Utilizing gold nanoparticles (Au-NPs) dual-functionalized with DNA and peptide, the presence and concentration of nuclease and protease are determined concurrently from the relative position and intensity of the bands in the staining-free gel electrophoresis. The use of Au-NPs eliminates the need for staining processes and enables naked eye detection, while a mononucleotide-mediated approach facilitates the synthesis of DNA/peptide conjugated Au-NPs and simplifies the operation procedures. Multiplex detection and quantification of DNase I and trypsin are successfully demonstrated.

Autoantibody detection by direct counting of antigen-displayed yeast cells.

In this study, we report a new immunoassay platform based on yeast surface display technology for detection of autoantibodies involved in autoimmune diseases, e.g., systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). The autoantigens of Ro52/SSA epitope and SmD were chosen to be displayed on the yeast surface with their respective antibodies as the analytes. By using magnetic beads modified with protein G, yeast cells bound with specific target antibody can be captured. The amount of analytes could be determined by counting the number of fluorescent yeast cells captured in a magnetic field. The platform showed promising results in the detection of SLE autoantibodies with high sensitivity and multiplex detection capability over the traditional approaches.

"Peak tracking chip" for label-free optical detection of bio-molecular interaction and bulk sensing.

A novel imaging method for bulk refractive index sensing or label-free bio-molecular interaction sensing is presented. This method is based on specially designed "Peak tracking chip" (PTC) involving "tracks" of adjacent resonant waveguide gratings (RWG) "micropads" with slowly evolving resonance position. Using a simple camera the spatial information robustly retrieves the diffraction efficiency, which in turn transduces either the refractive index of the liquids on the tracks or the effective thickness of an immobilized biological layer. Our intrinsically multiplex chip combines tunability and versatility advantages of dielectric guided wave biochips without the need of costly hyperspectral instrumentation. The current success of surface plasmon imaging techniques suggests that our chip proposal could leverage an untapped potential to routinely extend such techniques in a convenient and sturdy optical configuration toward, for instance for large analytes detection. PTC design and fabrication are discussed with challenging process to control micropads properties by varying their period (step of 2 nm) or their duty cycle through the groove width (steps of 4 nm). Through monochromatic imaging of our PTC, we present experimental demonstration of bulk index sensing on the range [1.33-1.47] and of surface biomolecule detection of molecular weight 30 kDa in aqueous solution using different surface densities. A sensitivity of the order of 10(-5) RIU for bulk detection and a sensitivity of the order of ∼10 pg mm(-2) for label-free surface detection are expected, therefore opening a large range of application of our chip based imaging technique. Exploiting and chip design, we expect as well our chip to open new direction for multispectral studies through imaging.