Sample-to-Answer Detection of SARS-CoV-2 Viremia Using Thermally Responsive Alkane Partitions.

The diagnosis of bloodborne viral infections (viremia) is currently relegated to central laboratories because of the complex procedures required to detect viruses in blood samples. The development of point-of-care diagnostics for viremia would enable patients to receive a diagnosis and begin treatment immediately instead of waiting days for results. Point-of-care systems for viremia have been limited by the challenges of integrating multiple precise steps into a fully automated (i.e., sample-to-answer), compact, low-cost system. We recently reported the development of thermally responsive alkane partitions (TRAPs), which enable the complete automation of diagnostic assays with complex samples. Here we report the use of TRAPs for the sample-to-answer detection of viruses in blood using a low-cost portable device and easily manufacturable cassettes. Specifically, we demonstrate the detection of SARS-CoV-2 in spiked blood samples, and we show that our system detects viremia in COVID-19 patient samples with good agreement to conventional RT-qPCR. We anticipate that our sample-to-answer system can be used to rapidly diagnose SARS-CoV-2 viremia at the point of care, leading to better health outcomes for patients with severe COVID-19 disease, and that our system can be applied to the diagnosis of other life-threatening bloodborne viral diseases, including Hepatitis C and HIV.

Saliva-STAT: Sample-to-answer saliva test for COVID-19.

Highly accessible and highly accurate diagnostics are necessary to combat rapidly-spreading infectious diseases, such as the recent COVID-19 pandemic. While lateral flow antigen tests have become pervasive, they are insufficiently sensitive to detect early or asymptomatic disease. Nucleic acid amplification tests provide the needed sensitivity, but accessibility of these tests continues to be a challenge due to the need for precise sample processing steps. Here we report a sample-to-answer test for saliva samples (saliva-STAT) that utilizes a battery-powered handheld instrument and a low-cost easily-manufacturable sample cassette to perform a nucleic acid amplification test for viral pathogens. To enable a completely automated assay, we leverage thermally responsive alkane partitions (TRAPs) and paramagnetic beads for virus purification and concentration, as well as reagent addition and mixing. Notably, the saliva STAT easily accommodates directly-dispensed saliva samples (in contrast to microfluidic devices), which is necessary for self-testing. Using the saliva-STAT platform, we demonstrate detection of down to 0.2 copies/µL of SARS-CoV-2 virus in saliva samples. We envision that the saliva-STAT could be used in walk-in clinics, mobile clinics, public testing locations, and in the home. With minor adjustments to the assay, the saliva-STAT platform can easily be adapted for other respiratory viruses, such as Influenza.

Effect of heterogeneity in recombination rate on variation in realised relationship.

Individuals of a specified pedigree relationship vary in the proportion of the genome they share identical by descent, i.e. in their realised or actual relationship. Predictions of the variance in realised relationship have previously been based solely on the proportion of the map length shared, which requires the implicit assumption that both recombination rate and genetic information are uniformly distributed along the genome. This ignores the possible existence of recombination hotspots, and fails to distinguish between coding and non-coding sequences. In this paper, we therefore quantify the effects of heterogeneity in recombination rate at broad and fine-scale levels on the variation in realised relationship. Variance is usually greater on a chromosome with a non-uniform recombination rate than on a chromosome with the same map length and uniform recombination rate, especially if recombination rates are higher towards chromosome ends. Reductions in variance can also be obtained, however, and the overall pattern of change is quite complex. In general, local (fine-scale) variation in recombination rate, e.g. hotspots, has a small influence on the variance in realised relationship. Differences in rates across longer regions and between chromosome ends can increase or decrease the variance in a realised relationship, depending on the genomic architecture.

Demonstration of a quantitative triplex LAMP assay with an improved probe-based readout for the detection of MRSA.

As molecular diagnostics move away from polymerase chain reaction (PCR) in order to target point-of-care testing applications, loop-mediated isothermal amplification (LAMP) is gaining popularity due its rapid, sensitive and specific detection with simpler instrumentation. However, while Taqman PCR enables real-time quantitative readout and multiplexed gene detection in single samples, analogous methods in LAMP are not yet broadly developed. To date, the real-time detection methods applied to LAMP involve turbidimetry or measuring fluorescence of an intercalator; however, both of these methods are nonspecific to the target of interest and do not allow for multiple gene detection in a single sample. Probe-based methods have been developed to address the need for specific target detection and multiplexed, one-pot reactions, but most of these methods have strict assay conditions and require the design of loop primers, which is not always possible. DARQ LAMP is a probe-based method that offers the most promise for quantitative and real-time multiplexed detection, as it has a relatively simple design and can be used in either a four-primer or six-primer system. However, previous work has only shown the assay to function well in a narrow range of reaction conditions, which is restrictive given that various LAMP assays require a broad range of conditions. In this work we investigate the use of the newest-generation strand-displacing polymerase and demonstrate that it has higher tolerance to reaction conditions than previous polymerases. Using the results from these studies, we demonstrate a single-reaction triplex assay for the detection of methicillin-resistant S. aureus (MRSA), which would not be possible with any of the previously reported LAMP systems.

Phase-Change Partitions for Thermal Automation of Multistep Reactions.

Medical diagnostics and basic research in low-resource settings require automated reactions to be controlled in a simple, portable manner. Here, we present a novel platform that enables simple automation of multistep reactions to facilitate robust, hands-free assay operation without complex microfluidics or paperfluidics. We separate reagent zones in a conventional PCR tube via solid layers of purified higher alkanes. Reagents can be mixed on demand by simply raising the temperature above the melting point of the alkane partition that separates the two zones. We partitioned various reagents to enable hands-free thermally automated isothermal nucleic acid amplification, heavy metal ion detection, and ß-lactamase detection with tandem antibiotic specificity characterization. We anticipate that this phase-change partition platform will find broad application in clinical diagnostics at the point-of-care and in low-resource settings.

Simplifying Nucleic Acid Amplification from Whole Blood with Direct Polymerase Chain Reaction on Chitosan Microparticles.

Tremendous advances have been made in the development of portable nucleic acid amplification devices for near-patient use. However, the true limitation in the realization of nucleic acid amplification tests (NAATs) for near-patient applications is not the amplification reaction, it is the complexity of the sample preparation. Conventional approaches require several precise intervention steps during the protocol. There are numerous reports in the literature that mimic the sample preparation procedure within a lab-on-a-chip device or cartridge, but these systems require a high number of integrated steps, making the devices and/or their supporting equipment too complex to meet the necessary cost targets and regulatory requirements for near-patient applications. Here we report a simplified method to purify and amplify DNA from complex samples in a minimal number of steps. We show that chitosan-coated microparticles can lyse human cells and capture the released DNA in a single mechanical agitation step, and we show that bound DNA can be amplified directly from the microparticle surface when the magnetic microparticles are transferred to a polymerase chain reaction (PCR). This procedure eliminates (i) the use of PCR-inhibiting reagents (e.g., chaotropic salts and alcohol) and (ii) the washing and elution steps that are required to remove these reagents and release DNA in typical NAAT sample preparation methods. To illustrate the use of this direct PCR method in diagnostics, we amplify human genomic DNA sequences from a ∼1 µL droplet of whole blood, and we amplify plasmid DNA spiked into whole blood droplets to represent circulating viral DNA or cell-free DNA. The qPCR threshold cycle for direct PCR from whole blood is comparable to that of direct PCR with purified DNA, demonstrating that the lysis and capture steps effectively bind DNA and sufficiently enable its amplification. Furthermore, the efficient amplification of plasmid DNA spiked into whole blood proves that the large mass of human genomic DNA captured from the lysed cells does not inhibit the capture and amplification of other circulating DNA. We anticipate that this new streamlined method for preparing DNA for amplification will expand the diagnostic applications of nucleic acid amplification tests, in particular for near-patient applications.

Peroxidyme-Amplified Radical Chain Reaction (PARCR): Visible Detection of a Catalytic Reporter.

Peroxidyme Amplified Radical Chain Reaction (PARCR), a novel enzyme-free system that achieves exponential amplification of a visible signal, is presented. Typical enzyme-free amplification systems that produce a visible readout suffer from long reaction times, low sensitivity, and narrow dynamic range. PARCR employs photocatalyzed nonlinear signal generation, enabling unprecedented one-pot, naked-eye detection of a catalytic reporter from 1 µm down to 100 pm. In this reaction, hemin-binding peroxidase-mimicking DNAzymes ("peroxidymes") mediate the NADH-driven oxidation of a colorless, nonfluorescent phenoxazine dye (Amplex Red) to a brightly colored, strongly fluorescent product (resorufin); illumination with green light initiates multiple radical-forming positive-feedback loops, rapidly producing visible levels of resorufin. Collectively, these results demonstrate the potential of PARCR as an easy-to-use readout for a range of detection schemes, including aptamer labels, hybridization assays, and nucleic acid amplification.

Capture and Direct Amplification of DNA on Chitosan Microparticles in a Single PCR-Optimal Solution.

While nucleic acid amplification tests have great potential as tools for rapid diagnostics, complicated sample preparation requirements inhibit their use in near-patient diagnostics and low-resource-setting applications. Recent advancements in nucleic acid purification have leveraged pH-modulated charge switching polymers to reduce the number of steps required for sample preparation. The polycation chitosan (pKa 6.4) has been used to efficiently purify DNA by binding nucleic acids in acidic buffers and then eluting them at a pH higher than 8.0. Though it is an improvement over conventional methods, this multistep procedure has not transformed the application of nucleic acid amplification assays. Here we describe a simpler approach using magnetic chitosan microparticles that interact with DNA in a manner that has not been reported before. The microparticles capture DNA at a pH optimal for PCR (8.5) just as efficiently as at low pH. Importantly, the captured DNA is still accessible by polymerase, enabling direct amplification from the microparticles. We demonstrate quantitative PCR from DNA captured on the microparticles, thus eliminating nearly all of the sample preparation steps. We anticipate that this new streamlined method for preparing DNA for amplification will greatly expand the diagnostic applications of nucleic acid amplification tests.

Higher phylogeny of frugivorous flies (Diptera, Tephritidae, Dacini): localised partition conflicts and a novel generic classification.

The phylogenetic relationships within and among subtribes of the fruit fly tribe Dacini (Ceratitidina, Dacina, Gastrozonina) were investigated by sequencing four mitochondrial and one nuclear gene fragment. Bayesian, maximum likelihood and maximum parsimony analyses were implemented on two datasets. The first, aiming at obtaining the strongest phylogenetic signal (yet, having lower taxon coverage), consisted of 98 vouchers and 2338 concatenated base pairs (bp). The second, aiming at obtaining the largest taxonomic coverage (yet, providing lower resolution), included 159 vouchers and 1200 concatenated bp. Phylogenetic relationships inferred by different tree reconstruction methods were largely congruent and showed a general agreement between concatenated tree topologies. Yet, local conflicts in phylogenetic signals evidenced a number of critical sectors in the phylogeny of Dacini fruit flies. All three Dacini subtribes were recovered as monophyletic. Yet, within the subtribe Ceratitidina only Perilampsis and Capparimyia formed well-resolved monophyletic groups while Ceratitis and Trirhithrum did not. Carpophthoromyia was paraphyletic because it included Trirhithrum demeyeri and Ceratitis connexa. Complex phylogenetic relationships and localised conflict in phylogenetic signals were observed within subtribe Dacina with (a) Dacus, (b) Bactrocera (Zeugodacus) and (c) all other Bactrocera species forming separate clades. The subgenus Bactrocera (Zeugodacus) is therefore raised to generic rank (Zeugodacus Hendel stat. nov.). Additionally, Bactrocera subgenera grouped under the Zeugodacus group should be considered under new generic combinations. Although there are indications that Zeugodacus and Dacus are sister groups, the exact relationship between Zeugodacus stat. nov., Dacus and Bactrocera still needs to be properly resolved.

Context-dependent redox properties of natural phenolic materials.

Macromolecular phenolics are among the most abundant organic molecules in nature, yet their biological activities are largely unresolved because of their structural complexity and because of an inability to probe their functionality experimentally. We developed thin film and electrochemical methodologies to probe the redox properties of melanin, lignin, and humic acid, three of the most abundant phenolic materials. We observed that all three phenolic matrixes possess redox activity and can be repeatedly switched between oxidized and reduced states. Furthermore, we observed that melanin possesses pro-oxidant activities exemplified by the uncatalyzed generation of reactive oxygen species (ROS) upon exposure to air; however, this pro-oxidant activity is observed only for melanin films that are poised in their reduced state. Conversely, melanin's antioxidant radical-scavenging activities are insensitive to its redox state. These results demonstrate that natural phenolic matrixes are not inert but rather serve as open-source redox media with significant potential for impacting redox signaling and redox biology.

Redox-capacitor to connect electrochemistry to redox-biology.

It is well-established that redox-reactions are integral to biology for energy harvesting (oxidative phosphorylation), immune defense (oxidative burst) and drug metabolism (phase I reactions), yet there is emerging evidence that redox may play broader roles in biology (e.g., redox signaling). A critical challenge is the need for tools that can probe biologically-relevant redox interactions simply, rapidly and without the need for a comprehensive suite of analytical methods. We propose that electrochemistry may provide such a tool. In this tutorial review, we describe recent studies with a redox-capacitor film that can serve as a bio-electrode interface that can accept, store and donate electrons from mediators commonly used in electrochemistry and also in biology. Specifically, we (i) describe the fabrication of this redox-capacitor from catechols and the polysaccharide chitosan, (ii) discuss the mechanistic basis for electron exchange, (iii) illustrate the properties of this redox-capacitor and its capabilities for promoting redox-communication between biology and electrodes, and (iv) suggest the potential for enlisting signal processing strategies to "extract" redox information. We believe these initial studies indicate broad possibilities for enlisting electrochemistry and signal processing to acquire "systems level" redox information from biology.

Highly sensitive and flexible inkjet printed SERS sensors on paper.

Surface enhanced Raman spectroscopy (SERS) has the potential to be utilized for the detection of a broad range of chemicals in trace quantities. However, because of the cost and complexity of SERS devices, the technology has been unable to fill the needs of many practical applications, in particular the need for rapid, portable, on-site detection in the field. In this work, we review a new methodology for trace chemical detection using inkjet-printed SERS substrates on paper. The detection performance of the inkjet-printed SERS devices is demonstrated by detecting 1,2-Bis(4-pyridyl)ethylene (BPE) at a concentration as low as 1.8 ppb. We then illustrate the primary advantages of paper SERS substrates as compared to conventional SERS substrates. By leveraging lateral flow concentration, the detection limit of paper SERS substrates can be further improved. Two real-world applications are demonstrated. First, the inkjet-printed SERS substrates are used as "dipsticks" for detecting the fungicide malachite green in water. Then, the flexible paper-based SERS devices are used as swabs to collect and detect trace residues of the fungicide thiram from a surface. We predict that the combination of ultra-low-cost fabrication with the advantages of easy-to-use dipsticks and swabs and the option of lateral flow concentration position ink-jet printed SERS substrates as a technology which will enable the application of SERS in solving critical problems for chemical detection in the field.

Simple SERS substrates: powerful, portable, and full of potential.

Surface enhanced Raman spectroscopy (SERS) is a powerful spectroscopic technique capable of detecting trace amounts of chemicals and identifying them based on their unique vibrational characteristics. While there are many complex methods for fabricating SERS substrates, there has been a recent shift towards the development of simple, low cost fabrication methods that can be performed in most labs or even in the field. The potential of SERS for widespread use will likely be realized only with development of cheaper, simpler methods. In this Perspective article we briefly review several of the more popular methods for SERS substrate fabrication, discuss the characteristics of simple SERS substrates, and examine several methods for producing simple SERS substrates. We highlight potential applications and future directions for simple SERS substrates, focusing on highly SERS active three-dimensional nanostructures fabricated by inkjet and screen printing and galvanic displacement for portable SERS analysis - an area that we believe has exciting potential for future research and commercialization.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive displacement assay is applicable to detection of a variety of biological macromolecules, including proteins and proteolytic enzymes.

Enrichment of tumor-initiating breast cancer cells within a mammosphere-culture microdevice.

We report for the first time a microdevice that enables the selective enrichment, culture, and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade, researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However, the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, such as cell sorting, drug screens, and molecular assays. In this work, we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres, it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device, the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane, and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability, we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity, a cancer stem cell marker. To the best of our knowledge, this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere.

Inkjet-printed paper-based SERS dipsticks and swabs for trace chemical detection.

We demonstrate a paper-based surface swab and lateral-flow dipstick that includes an inkjet-printed surface-enhanced Raman spectroscopy (SERS) substrate for analyte detection. Due to capillary-action wicking of cellulose, the paper dipstick enables extremely simple and pump-free loading of liquid samples into the detection device, and in addition provides inherent analyte concentration within the detection volume. Furthermore, the flexible nature of the paper-based SERS device also enables it to act as a swab to collect analyte molecules directly from a large-area surface; the collected analyte molecules can then be focused into a small-volume SERS-active region by lateral-flow concentration. These capabilities are unseen in today's SERS substrates and microfluidic SERS devices. Using these novel lateral-flow paper SERS devices, we achieved detection limits as low as 95 fg of Rhodamine 6G (R6G), 413 pg of the organophosphate malathion, 9 ng of heroin, and 15 ng of cocaine. Moreover, the measurements show that the technique is quantitative and is repeatable across multiple swabs and dipsticks. The results reported here may lead to ultra-low-cost portable applications in trace chemical detection.

Multiplexed detection of aquaculture fungicides using a pump-free optofluidic SERS microsystem.

In this work, an optofluidic SERS device optimized for on-site analytics in the field is utilized for the multiplexed detection of three fungicides that are highly regulated in aquaculture. The optofluidic SERS microsystem does not require a bulky pump for sample loading, which significantly improves its portability; the sample is simply loaded into the device by applying negative pressure using a pipette. Moreover, integrated fiber optic cables automate sample excitation and signal collection without the need for alignment on a traditional Raman microscope. The detection zone of the device consists of a porous matrix of packed silica microspheres that accumulates silver nanoparticles and adsorbed analyte molecules. This passive concentration matrix has been shown to boost the SERS signal by up to four orders of magnitude as compared to SERS in an open microfluidic channel. We were able to detect as low as 5 ppm methyl parathion, 0.1 ppb malachite green, and 5 ppb thiram simultaneously.

Chromatographic separation and detection of target analytes from complex samples using inkjet printed SERS substrates.

In principle, surface enhanced Raman spectroscopy (SERS) is thought to provide unique identification of a target analyte, even in complex samples or in the presence of multiple analytes. In practice, however, this is not always true for real-world samples due to various forms of interference. In this report, we build upon our previous work on inkjet-printed SERS substrates by using paper and polymer membranes to integrate sample cleanup and analyte separation with SERS detection. Inkjet-printed paper SERS substrates provide a highly sensitive chemical detection platform of unprecedented cost and simplicity. In addition, paper inherently provides unique capabilities, such as capillary-actuated fluid transport and selective molecular retention. Utilizing these properties, we demonstrate two-dimensional chromatographic separation and SERS detection on inkjet-printed paper SERS substrates. Then, we leverage the separation properties of paper and polymer membranes for real applications that feature complex sample matrices, including the detection of down to 5 ppm melamine in infant formula, as well as the quantification of nanograms of heroin in samples contaminated with a highly fluorescent background. The results presented here demonstrate that inkjet-printed paper SERS devices not only provide advantages in terms of sensitivity and cost, but the paper provides inherently integrated sample cleanup capabilities that are not available in traditional SERS substrates and microfluidic SERS devices. These unique capabilities of paper SERS devices enable the identification of targeted analytes even in complex real-world samples.

Optofluidic surface enhanced Raman spectroscopy microsystem for sensitive and repeatable on-site detection of chemical contaminants.

We demonstrate highly sensitive detection of real-world food and water contaminants using a portable and automated optofluidic surface enhanced Raman spectroscopy (SERS) microsystem. The optofluidic SERS device utilizes a porous microfluidic matrix formed by packed silica microspheres to concentrate silver nanoparticles and adsorbed analyte molecules, resulting in greatly improved SERS detection performance. In addition, a passive micromixer that mixes silver nanoparticles into the sample solution is integrated into the device for improved automation. Furthermore, two optical fibers are integrated into the device and aligned to the detection volume to improve the automation as compared to confocal SERS, which requires focusing and alignment. The device exhibits up to 2 orders of magnitude improvement in SERS performance as compared to conventional microfluidic SERS in an open channel. Using the optofluidic SERS device, the food contaminant melamine was detected in low concentrations, with an estimated limit of detection (LOD) of 63 ppb, while the fungicide thiram was detected down to an estimated LOD of 50 ppt. In both cases, the reported results meet the U.S. federal requirements. Additionally, it is shown that the device continues to exhibit excellent performance even when mated to a commercially available portable spectrometer for the trace detection of thiram. This combination of the optofluidic SERS microsystem with a portable spectrometer will lead to highly sensitive and automated sensing systems for on-site detection of food and water contaminants in the field.

A simple filter-based approach to surface enhanced Raman spectroscopy for trace chemical detection.

We demonstrate an extremely simple and practical surface enhanced Raman spectroscopy (SERS) technique for trace chemical detection. Filter membranes first trap silver nanoparticles to form a SERS-active substrate and then concentrate analytes from a mL-scale sample into a µL-scale detection volume. We demonstrate a significant improvement in detection limit as compared to colloidal SERS for the pesticide malathion and the food contaminant melamine. The measured SERS intensity exhibits low variation relative to traditional SERS techniques, and the data can be closely fit with a Langmuir isotherm. Thus, due to the simple procedure, the low-cost of the substrates, the quantitative results, and the performance improvement due to analyte concentration, our technique enables SERS to be practical for a broad range of analytical applications, including field-based detection of toxins in large-volume samples.