Sample-to-Answer Immuno-Magnetic Assay Using Thermally Responsive Alkane Partitions.

To combat pandemics, there is a need for rapid point-of-care diagnostics to identify infected patients and to track the spread of the disease. While recent progress has been made in response to COVID-19, there continues to be a need for point-of-care diagnostics capable of detecting biomarkers-such as antibodies-in whole blood. We have recently reported the development of thermally responsive alkane partitions (TRAPs) for the automation of point-of-care immuno-magnetic assays. Here, we demonstrate the use of TRAPs to enable sample-to-answer detection of antibodies against the SARS-CoV-2 virus in whole blood samples. We report a limit of detection of 84 pg/mL, well below the clinically relevant threshold. We anticipate that the TRAP-enabled sample-to-answer immunoassay can be used to track the progression of future pandemics, leading to a more informed and robust clinical and societal response.

Thermally Responsive Alkane Partitions for Assay Automation.

For point-of-care diagnostic tools to be impactful, they must be inexpensive, equipment-free, and sample-to-answer (i.e., require no user intervention). Here, we report a new approach to enable sample-to-answer diagnostics that utilizes thermally responsive alkane partitions (TRAPs) as automated pseudo-valves. When combined with the magnetic manipulation of microbeads, TRAPs enable the pumpless automation of all steps in complex assays. We demonstrate that in relatively narrow channel geometries, liquified alkane partitions continue to separate reagents on each side of the partition while enabling the transition of magnetic beads from one reagent to the next, replacing manual pipetting steps in conventional assays. In addition, we show that in relatively broader geometries, liquified partitions breach, enabling the addition/mixing of preloaded reagents. Through calculation and experimentation, we determine the geometric design rules for implementing the stationary and removable partitions in fluidic channels. In addition, we demonstrate that magnetic microbeads can be pulled through liquified stationary TRAPs without disrupting partition integrity and without disrupting bound protein complexes attached at the microbead surface. The TRAP technology introduced here can enable a new low-cost and equipment-free approach for fully automated sample-to-answer diagnostics.

Sample-to-Answer Diagnostic System for the Detection of Circulating Histones in Whole Blood.

Severe internal trauma results in millions of hospitalizations each year, including thousands of deaths caused by subsequent multiple organ failure. The majority of these deaths occur within the first 24 h, and thus, rapid diagnosis of internal trauma severity is necessary for immediate treatment. For early organ damage identification, diagnosis in point-of-care settings is crucial for rapid triage and treatment. Recent reports suggest that circulating histones may serve as a biomarker for severe organ damage and the risk of multiple organ failure. Here, we report a point-of-care diagnostic system that utilizes the inherent interactions between histones and DNA for the fluorescence-based detection of histones in whole blood. In the assay, histones within the sample are wrapped by DNA, thus preventing an intercalating dye from binding the DNA and fluorescing. To allow for quantitative fluorescent measurements to be made in a point-of-care setting, we integrate a rapid, automated blood separation step into our assay. Furthermore, we eliminate manual reagent additions using a thermally responsive alkane partition (TRAP), thus making the system sample-to-answer. Finally, we demonstrate the assay in a portable fluorescence reader compatible with a point-of-care environment. We report a limit of detection 112 ng/mL in whole blood, suggesting that our device can be used to rapidly diagnose internal trauma severity and the likelihood of multiple organ failure in near-patient settings.

A critical review of point-of-care diagnostic technologies to combat viral pandemics.

The COVID-19 global pandemic of 2019-2020 pointedly revealed the lack of diagnostic solutions that are able to keep pace with the rapid spread of the virus. Despite the promise of decades of lab-on-a-chip research, no commercial products were available to deliver rapid results or enable testing in the field at the onset of the pandemic. In this critical review, we assess the current state of progress on the development of point-of-care technologies for the diagnosis of viral diseases that cause pandemics. While many previous reviews have reported on progress in various lab-on-a-chip technologies, here we address the literature from the perspective of the testing needs of a rapidly expanding pandemic. First, we recommend a set of requirements to heed when designing point-of-care diagnostic technologies to address the testing needs of a pandemic. We then review the current state of assay technologies with a focus on isothermal amplification and lateral-flow immunoassays. Though there is much progress on assay development, we argue that the largest roadblock to deployment exists in sample preparation. We summarize current approaches to automate sample preparation and discuss both the progress and shortcomings of these developments. Finally, we provide our recommendations to the field of specific challenges to address in order to prepare for the next pandemic.

Advances in the analysis of single extracellular vesicles: A critical review.

There is an ever-growing need for new cancer diagnostic approaches that provide earlier diagnosis as well as richer diagnostic, prognostic, and resistance information. Extracellular vesicles (EVs) recovered from a liquid biopsy have paradigm-shifting potential to offer earlier and more complete diagnostic information in the form of a minimally invasive liquid biopsy. However, much remains unknown about EVs, and current analytical approaches are unable to provide precise information about the contents and source of EVs. New approaches have emerged to analyze EVs at the single particle level, providing the opportunity to study biogenesis, correlate markers for higher specificity, and connect EV cargo with the source or destination. In this critical review we describe and analyze methods for single EV analysis that have emerged over the last five years. In addition, we note that current methods are limited in their adoption due to cost and complexity and we offer opportunities for the research community to address this challenge.

Phenotypically distinguishing ESBL-producing pathogens using paper-based surface enhanced Raman sensors.

Antimicrobial stewardship practices are critical in preventing the further erosion of treatment options for bacterial infections. Yet, at the same time, determination of an infection's antimicrobial susceptibility requires multiple rounds of culture and expensive lab automation systems. In this work, we report the use of paper-based surface enhanced Raman spectroscopy (SERS) sensors and portable instrumentation to phenotypically discriminate multi-drug resistance with fewer culture steps than conventional clinical microbiology. Specifically, we demonstrate the identification of resistance to varying generations of ß-lactam antibiotics by detecting the activity of particular ß-lactamase enzymes in a multiplexed assay. The method utilizes molecular reporters that consist of ß-lactams with SERS barcodes. Hydrolysis of the ß-lactam by ß-lactamase enzymes in the sample expels the barcode; the released sulfur-containing barcode is then detected via SERS. Using this approach, we demonstrate the differentiation of E. coli strains with (1) extended spectrum ß-lactamase (ESBL), (2) narrow-spectrum ß-lactamase, and (3) no resistance, using only a single measurement on a single sample. In addition, we experimentally validate an approach to expand the library of reporters through the simple chemical synthesis of new barcoded ß-lactams. Importantly, the reported method determines the susceptibility based on phenotypic ß-lactamase activity, which is aligned with current microbiology lab standards. This new method will enable the precise selection of effective ß-lactam antibiotics (as opposed to defaulting to drugs of last resort) faster than current methods while using simple steps and low-cost portable instrumentation.

Nucleic acid-cleaving catalytic DNA for sensing and therapeutics.

DNAzymes with nucleic acid-cleaving catalytic activity are increasing in versatility through concerted efforts to discover new sequences with unique functions, and they are generating excitement in the sensing community as cheap, stable, amplifiable detection elements. This review provides a comprehensive list and detailed descriptions of the DNAzymes identified to date, classified by their associated small molecule or ion needed for catalysis; of note, this classification clarifies conserved regions of various DNAzymes that are not obvious in the literature. Furthermore, we detail the breadth of functionality of these DNA sequences as well as the range of reaction conditions under which they are useful. In addition, the utility of the DNAzymes in a variety of sensing and therapeutic applications is presented, detailing both their advantages and disadvantages.

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.

New Trimodal Phenotypic Reporter of Extended-Spectrum ß-Lactamase Activity.

Bacterial resistance to ß-lactam antibiotics continues to grow as misadministration presents evolutionary pressure that drives bacteria to develop improved resistance enzymes. Known as extended-spectrum ß-lactamases (ESBLs), these enzymes are capable of hydrolyzing advanced ß-lactam antibiotics such as third-generation (and higher) cephalosporins. Phenotypic detection substrates can be used to rapidly identify a cultured patient sample prior to confirmation by more exhaustive but slower means, critically aiding in the antibiotic stewardship essential in maintaining the effectiveness of not only the cephalosporins but also indirectly the carbapenems, our last-resort ß-lactams. To enhance the phenotypic detection arsenal, we have designed an ESBL detection substrate that releases a glucose molecule upon ß-lactamase hydrolysis. Because many forms of detection for glucose exist, the substrate enables ESBL quantification via three modalities commonly found in the clinical laboratory: optical absorbance, for use with the most common microbiology platforms; fluorescence, for enhanced sensitivity; and electrochemistry, which offers the potential for integration into a hand-held platform similar to a personal glucometer. Moreover, we demonstrate that, as opposed to currently available phenotypic detection substrates, our new substrate is engineered to be resistant to older and narrower ß-lactamases, thus enabling specific identification of newer and more dangerous ESBLs.

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.

A critical review of flexible and porous SERS sensors for analytical chemistry at the point-of-sample.

For decades surface enhanced Raman spectroscopy (SERS) has been intensely investigated as a possible solution for performing analytical chemistry at the point of sample origin. Unfortunately, due to cost and usability constraints, conventional rigid SERS sensors and microfluidic SERS sensors have yet to make a significant impact outside of the realm of academics. However, the recently introduced flexible and porous paper-based SERS sensors are proving to be widely adaptable to realistic usage cases in the field. In contrast to rigid and microfluidic SERS sensors, paper SERS sensors feature (i) the potential for roll-to-roll manufacturing methods that enable low sensor cost, (ii) simple sample collection directly onto the sensor via swabbing or dipping, and (iii) equipment-free separations for sample cleanup. In this review we argue that movement to paper-based SERS sensors will finally enable point-of-sample analytical chemistry applications. In addition, we present and compare the numerous fabrication techniques for paper SERS sensors and we describe various sample collection and sample clean-up capabilities of paper SERS sensors, with a focus on how these features enable practical applications in the field. Finally, we present our expectations for the future, including emerging ideas inspired by paper SERS.

Multistage Chemical Heating for Instrument-Free Biosensing.

Improving the portability of diagnostic medicine is crucial for alleviating global access-to-care deficiencies. This requires not only designing devices that are small and lightweight, but also autonomous and independent of electricity. Here, we present a strategy for conducting automated multistep diagnostic assays using chemically generated, passively regulated heat. Ligation and polymerization reagents for rolling circle amplification of nucleic acids are separated by meltable phase-change partitions, thus replacing precise manual reagent additions with automated partition melting. To actuate these barriers and individually initiate the various steps of the reaction, field ration heaters exothermically generate heat in a thermos, whereas fatty acids embedded in a carbonaceous matrix passively buffer the temperature around their melting points. Achieving multistage temperature profiles extend the capability of instrument-free diagnostic devices and improve the portability of reaction automation systems built around phase-change partitions.

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.

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.

Inkjet-Printed Paper Fluidic Devices for Onsite Detection of Antibiotics Using Surface-Enhanced Raman Spectroscopy.

Surface enhanced Raman spectroscopy (SERS) provides rapid and sensitive identification of small molecule analytes. Traditionally, fabrication of SERS devices is an expensive process that involves the use of micro- and nano-fabrication procedures. Further, acquisition of diverse sample types requires complex preparation procedures that limits SERS to lab-based applications. Recent innovations using plasmonic nanoparticles embedded in flexible paper substrates has allowed the expansion of SERS techniques to portable analytical procedures. Recently inkjet-printing has been identified as a low cost, rapid, and highly customizable method for producing paper based SERS sensors with robust performance. This chapter details the materials and procedures by which inkjet printed SERS sensors can be fabricated and applied to relevant applications. In particular, methods for utilizing the sensors for detection of antibiotics are presented.

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.

Vertical-flow paper SERS system for therapeutic drug monitoring of flucytosine in serum.

A number of life-saving drugs require therapeutic drug monitoring (TDM) for safe and effective use. Currently, however, TDM is performed using sophisticated analytical techniques relegated to central labs, increasing the cost per test and time to answer. Here, using a novel vertical flow membrane system with inkjet-printed surface enhanced Raman sensors, along with a portable spectrometer, we demonstrate a low cost and easy to use device to quantify levels of flucytosine, an antifungal that requires TDM for effective patient care, from undiluted human serum. To our knowledge, this work represents the first report of a passive vertical flow sample cleanup method with surface enhanced Raman detection. We first investigated and optimized the parameters of the vertical flow system for the detection of flucytosine in spiked serum samples. Then, using an optimized vertical-flow system utilizing nitrocellulose membranes and a paper SERS sensor, we achieved detection of down to 10 µg mL-1 flucytosine in undiluted serum, with quantitative detection across the entire therapeutic range. This system reduces the assay time to about 15 min, far quicker than the current gold standards. We anticipate that this novel system will enable near-patient therapeutic drug monitoring, leading to the safe and effective administration of a number of life-saving drugs. Furthermore, it will spawn the development of SERS detection systems capable of separating target analytes from real-world biological matrices.

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.

Assessment of surfactants for efficient droplet PCR in mineral oil using the pendant drop technique.

Amplification and detection of nucleic acid sequences within integrated microsystems is routinely conducted using the technique of droplet PCR, wherein the polymerase chain reaction (PCR) is performed in microscale water-in-oil droplets (nanoliter to picoliter volumes). During droplet PCR, interactions at the interface of the droplet tend to dominate. Specifically, adsorption of the polymerase at the droplet interface leads to inefficient amplification. To reduce polymerase adsorption, surfactants such as the silicone-based ABIL EM90 have been commonly used. However, these surfactants have been selected largely through trial and error, and have been only somewhat effective. For example, when using ABIL EM90, 8 times (8 ×) the manufacturer prescribed concentration of polymerase was necessary for amplification. In this report, we use the pendant drop technique to measure adsorption and loss of enzyme at droplet interfaces for various surfactant-oil combinations. Dynamic interfacial tension and surface pressure measurements showed that significant polymerase adsorption occurs when using ABIL EM90. In contrast, much lower polymerase adsorption is observed when using Brij L4, a nonionic surfactant with a C12 tail and an oxyethylene headgroup, which has not yet been reported for droplet PCR. These results correlate strongly with droplet PCR efficiency. Brij L4 enables highly efficient PCR at 2 × polymerase concentration, and still enables effective PCR at 1 × polymerase concentration. Overall, this work introduces a methodology for quantitatively assessing surfactants for use with droplet microreactors, and it demonstrates the practical value of this new approach by identifying a surfactant that can dramatically improve the efficiency of droplet PCR.