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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.

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., Graphical abstract Image 1, Highlights • Despite decades of research, no products can to meet the testing needs of a pandemic. • We propose new guidelines for the design of point-of-care tests for pandemics. • We summarize the literature on point-of-care diagnostics for pandemics. • We find that sample preparation steps are the primary hindrance to deployment. • We provide a list of recommended research topics to prepare for future pandemics.

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.

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

Bacterial resistance to β-lactam antibiotics continues to grow as misadministration presents evolutionary pressure that drives bacteria to develop improved resistance enzymes. Known as extended-spectrum β-lactamases (ESBLs), these enzymes are capable of hydrolyzing advanced β-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 β-lactams. To enhance the phenotypic detection arsenal, we have designed an ESBL detection substrate that releases a glucose molecule upon β-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 β-lactamases, thus enabling specific identification of newer and more dangerous ESBLs.

We present an experimental demonstration of the feasibility of the first 20 + Mb/s Gaussian modulated coherent state continuous variable quantum key distribution system with a locally generated local oscillator at the receiver (LLO-CVQKD). To increase the signal repetition rate, and hence the potential secure key rate, we equip our system with high-performance, wideband devices and design the components to support high repetition rate operation. We have successfully trialed the signal repetition rate as high as 500 MHz. To reduce the system complexity and correct for any phase shift during transmission, reference pulses are interleaved with quantum signals at Alice. Customized monitoring software has been developed, allowing all parameters to be controlled in real-time without any physical setup modification. We introduce a system-level noise model analysis at high bandwidth and propose a new ‘combined-optimization’ technique to optimize system parameters simultaneously to high precision. We use the measured excess noise, to predict that the system is capable of realizing a record 26.9 Mb/s key generation in the asymptotic regime over a 15 km signal mode fibre. We further demonstrate the potential for an even faster implementation.

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 β-lactam antibiotics by detecting the activity of particular β-lactamase enzymes in a multiplexed assay. The method utilizes molecular reporters that consist of β-lactams with SERS barcodes. Hydrolysis of the β-lactam by β-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 β-lactamase (ESBL), (2) narrow-spectrum β-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 β-lactams. Importantly, the reported method determines the susceptibility based on phenotypic β-lactamase activity, which is aligned with current microbiology lab standards. This new method will enable the precise selection of effective β-lactam antibiotics (as opposed to defaulting to drugs of last resort) faster than current methods while using simple steps and low-cost portable instrumentation.

Aqueous capsules made from polymers typically allow their encapsulated cargo (e.g., drugs, dyes, proteins) to slowly diffuse out into the solvent through the capsule shell. In many applications, there is a need for a ‘hermetic’ seal to protect the cargo from the solvent over extended periods of time. Ideally, this hermetic seal should also be capable of being broken on-demand to enable cargo release. We demonstrate a new design for capsules having the above combination of properties. The key is to create the capsule shell from wax materials (alkanes and fatty acids) with a defined melting temperature Tm. Capsules can be loaded with any desired material, including strong acids or bases, reactive or unstable reagents (such as H2O2), and biopolymer gels. When sealed capsules are placed in water, no leakage is observed for over six weeks. The capsules can also encapsulate volatile liquids and remain air-tight over at least three weeks. On the other hand, under mild heat (above Tm, e.g., to 45 °C), the shell melts, releasing the core contents into the surrounding solvent. This provides a convenient thermal on–off “switch” for delivering contents from the capsules. The utility of these capsules is shown by implementing a nitrate-detection assay using hazardous chemicals (including H2SO4) sealed in the capsules. These “smart” capsules thus constitute a modular, mass-producible platform that could be useful in diverse applications.

Most species attack the seed bearing organs of plants, including soft fruits and flowers. The taxonomy of the Tephritidae, or the true fruit flies, has applications in pest management and plant quarantine, weed biocontrol, and evolutionary biology. In the Oriental fruit fly species complex, about 60 sibling species are recognized so far, most of which are of limited distribution. The frequency of fruit fly symposia and the attendance they attract indicate that many hundreds of entomologists worldwide must be involved with the group of flies. A proportion of these entomologists work in areas with a high diversity of fruit flies or in areas with specific quarantine threats from areas with a diversity of species. Clearly there is a need for non-taxonomists to do a proportion of their own identification work, and this need has been addressed through training and the compilation of a general guide to pest fruit flies.

Sample filling and discretization within thermoplastic 2D microwell arrays is investigated toward the development of low cost disposable microfluidics for passive sample discretization. By using a high level of contact angle asymmetry between the filling channel and microwell surfaces, a significant increase in the range of well geometries that can be successfully filled is revealed. The performance of various array designs is characterized numerically and experimentally to assess the impact of contact angle asymmetry and device geometry on sample filling and discretization, resulting in guidelines to ensure robust microwell filling and sample isolation over a wide range of well dimensions. Using the developed design rules, reliable and bubble-free sample filling and discretization is achieved in designs with critical dimensions ranging from 20 μm to 800 μm. The resulting devices are demonstrated for discretized nucleic acid amplification by performing loop-mediated isothermal amplification for the detection of the mecA gene associated with methicillin-resistant Staphylococcus aureus.

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.

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.

The seamless integration of reagents into microfluidic devices can serve to significantly reduce assay complexity and cost for disposable diagnostics. In this work, the integration of multiplexed reagents into thermoplastic 2D microwell arrays is demonstrated using a scalable pin spotting technique. Using a simple and low-cost narrow-bore capillary spotting pin, high resolution deposition of concentrated reagents within the arrays of enclosed nanoliter-scale wells is achieved. The pin spotting method is further employed to encapsulate the deposited reagents with a chemically modified wax layer that serves to prevent disruption of the dried assay components during sample introduction through a shared microchannel, while also enabling temperature-controlled release after sample filling is complete. This approach supports the arbitrary patterning and release of different reagents within individual wells without crosstalk for multiplexed analyses. The performance of the in-well spotting technique is characterized using on-chip rolling circle amplification to evaluate its potential for nucleic acid-based diagnostics.

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.

Meltable barriers are an attractive means to achieve controlled delivery of reagents in a variety of settings, enabling assays to be performed through thermal automation instead of manual addition of reactants. However, mixing kinetics in such systems can be slow due to the lack of active flow or mechanical shaking. We demonstrate a new strategy for hands-free, thermally-automated agitation of biochemical reactions. Reagents for binary effervescent reactions are lyophilized then capped with a phase-change partition, eicosane. This barrier can be melted at moderate temperatures, at which point an aqueous solution dissolves the reactants, generating bubbles that mix the solution through convection. We explore reactions that generate bubbles of carbon dioxide and oxygen gasses, characterizing the induced mixing rate of two aqueous solutions with dissimilar densities. This strategy affords control over the initiation and duration of convective mixing, providing a tool for thermal automation of biochemical reactions with efficient reaction kinetics.

Improving the portability of diagnostic medicine is crucial to 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 multi-step diagnostic assays using chemically generated, passively regulated heat. Ligation and polymerization reagents for Rolling Circle Amplification of nucleic acids are separated by melt-able 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 while fatty acids embedded in a carbonaceous matrix passively buffer the temperature around their melting points. Achieving multi-stage temperature profiles extends the capability of instrument-free diagnostic devices and improves the portability of reaction automation systems built around phase-change partitions.

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 β-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.

Surface enhanced Raman spectroscopy (SERS) has the potential to enable point-of-care sensing across the spectrum of chemical and biological analytes. In diagnostic assays, SERS has been demonstrated to increase the multiplexing density while reducing the burden of fluorescence hardware. One particular application of interest is the use of SERS to provide a multiplexed optical read-out following polymerase chain reaction (PCR). To date, however, the reported PCR–SERS assays require endpoint mixing with a plasmonic nanoparticle solution for detection, thus adding manual steps and preventing real-time, quantitative PCR. In this work, we detail a real-time PCR–SERS thermoplastic microsystem that allows simultaneous nucleic acid amplification and product separation into a SERS-active silver colloid for real-time detection. Specifically, a laser cut thermoplastic fluidic chip has been devised to utilize a dialysis membrane capable of isolating a PCR reaction from the silver colloid. As the reaction progresses, a Raman-reporter-labeled DNA probe is degraded, liberating the reporter from probe DNA, allowing passage across the size-restricting dialysis membrane into the SERS-active colloid, where the accumulating reporter can be measured in real time. Here, we demonstrate that this system is capable of real-time and single-well multiplexed readout of a PCR reaction to simultaneously detect two biomarker genes for methicillin-resistant S. aureus (MRSA).

The purpose of this application, under Article 23.9.3, is to conserve current usage of the well-established genus-group name Cryptodacus Hendel, 1914 for a genus of Neotropical fruit flies by suppression of the earlier, unused name Cryptodacus Gundlach, 1862, currently a junior synonym of Arrhyton Gunther, 1858, a genus of snakes, under the plenary power of the Commission, in the interest of nomenclatural stability. Cryptodacus Gundlach has not been used as a valid name since 1883, whereas Cryptodacus Hendel has been used in a significant body of literature relating to fruit fly systematics, morphology and phylogeny and is the currently used name in various name and molecular databases.

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.

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.

A host choice experiment was carried out to determine the host range of some potential biological control agents of yellow starthistle (Centaurea solstitialis), a Mediterranean plant which has become a serious rangeland weed in western USA. This paper describes the results of that experiment and discusses its taxonomic implications. The experiment confirmed the extreme host specificity of some species. However, two reputedly polyphagous species, Acanthiophilus helianthi (Rossi) and Urophora quadrifasciata (Meigen), attacked a narrower range of hosts than expected. The results of this and similar host choice experiments are discussed and it is concluded that this sort of anomaly can be caused by the existence of host races, unrecognized sibling species, or by varieties of plants naturally resistant to attack. These complex patterns of host relationship highlight the need to interpret host catalogue data cautiously, as they oversimplify complex biological systems. The identity of one species reared in the experiment, Terellia luteola (Wiedemann), is discussed; a key is presented for its separation from other Terellia species, and it is removed from synonymy with T. colon (Meigen).

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.

This Conference Presentation was recorded at SPIE Photonics West 2017 held in San Francisco, California, United States.

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.

Surface Enhanced Raman spectroscopy (SERS) provides significant improvements over conventional methods for single and multianalyte quantification. Specifically, the spectroscopic fingerprint provided by Raman scattering allows for a direct multiplexing potential far beyond that of fluorescence and colorimetry. Additionally, SERS generates a comparatively low financial and spatial footprint compared with common fluorescence based systems. Despite the advantages of SERS, it has remained largely an academic pursuit. In the field of biosensing, techniques to apply SERS to molecular diagnostics are constantly under development but, most often, assay protocols are redesigned around the use of SERS as a quantification method and ultimately complicate existing protocols. Our group has sought to rethink common SERS methodologies in order to produce translational technologies capable of allowing SERS to compete in the evolving, yet often inflexible biosensing field. This work will discuss the development of two techniques for quantification of microRNA, a promising biomarker for homeostatic and disease conditions ranging from cancer to HIV. First, an inkjet-printed paper SERS sensor has been developed to allow on-demand production of a customizable and multiplexable single-step lateral flow assay for miRNA quantification. Second, as miRNA concentrations commonly exist in relatively low concentrations, amplification methods (e.g. PCR) are therefore required to facilitate quantification. This work presents a novel miRNA assay alongside a novel technique for quantification of nuclease driven nucleic acid amplification strategies that will allow SERS to be used directly with common amplification strategies for quantification of miRNA and other nucleic acid biomarkers.

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.

While surface enhanced Raman spectroscopy (SERS) may not compete with the standard central lab approaches for chemical and biological sensing, SERS may have the potential to provide unique capabilities for analytics away from the central lab. Raman spectrometers have evolved from benchtop systems to high-performing handheld instruments that are compatible with analysis of samples in the field. However, for SERS to truly succeed as a “point-of-sample” analytical technique, the SERS sensor must fit the needs of analysis in the field, including little or no sample preparation, minimal peripheral equipment, and ease of use. Traditional plasmonically-active rigid devices do not meet these requirements. Even microfluidic SERS devices generally are not compatible with point-of-sample analysis, as the "world-to-chip" interface presents challenges, and peripheral equipment is generally required. In this review we will discuss the advances in plasmonic substrates fabricated on porous membranes, leading to SERS sensors that can collect samples via swabbing or dipping, clean up samples through separation, concentrate analytes by lateral flow focusing, and avoid the need for peripheral equipment. In particular, we will focus on inkjet-fabricated devices, which may present the best opportunity for scale-up via roll-to-roll manufacturing. We will also discuss the directions that flexible SERS sensors are moving the field, such as simple fabrication techniques, new support materials, SERS swabs, and SERS-active tapes and films.

A need exists for near real-time therapeutic drug monitoring (TDM), in particular for antibiotics and antifungals in patient samples at the point-of-care. To truly fit the point-of-care need, techniques must be rapid and easy to use. Here we report a membrane system utilizing inkjet-fabricated surface enhanced Raman spectroscopy (SERS) sensors that allows sensitive and specific analysis despite the elimination of sophisticated chromatography equipment, expensive analytical instruments, and other systems relegated to the central lab. We utilize inkjet-fabricated paper SERS sensors as substrates for 5FC detection; the use of paper-based SERS substrates leverages the natural wicking ability and filtering properties of microporous membranes. We investigate the use of microporous membranes in the vertical flow assay to allow separation of the flucytosine from whole blood. The passive vertical flow assay serves as a valuable method for physical separation of target analytes from complex biological matrices. This work further establishes a platform for easy, sensitive, and specific TDM of 5FC from whole blood.

Surface-enhanced Raman spectroscopy (SERS) enables multiplex detection of analytes using simple, portable equipment consisting of a single excitation source and detector. Thus, in theory, SERS is ideally suited to replace fluorescence in assays that screen for numerous deoxyribonucleic acid (DNA) targets, but in practice, SERS-based assays have suffered from complexity and elaborate processing steps. Here, we report an assay in which a simple inkjetfabricated plasmonic paper device enables SERS-based detection of multiple DNA targets within a single polymerase chain reaction (PCR). In prior work, we demonstrated the principles of chromatographic separation and SERS-based detection on inkjet-fabricated plasmonic paper. The present work extends that capability for post-PCR gene sequence detection. In this design, hydrolysis DNA probes with 5′ Raman labels are utilized; if the target is present, the probe is hydrolyzed during PCR, freeing the reporter. After applying the PCR sample to a paper SERS device, an on-device chromatographic separation and concentration is conducted to discriminate between hydrolyzed and intact probes. SERS is then used to detect the reporter released by the hydrolyzed probes. This simple separation and detection on paper eliminates the need for complex sample processing steps. In this work, we simultaneously detect the methicillin-resistant Staphylococcus aureus genes mecA and femB to illustrate the concept. We envision that this approach could contribute to the development of multiplex DNA diagnostic tests enabling screening for several target sequences within a single reaction, which is necessary for cases in which sample volume and resources are limited.

Tephritid fruit flies, or "true" fruit flies (Diptera, Tephritidae) include approximately 500 genera and 4800 valid species (Norrbom 2004), whose vast majority (95%) is represented by phytophagous species (reviewed in Aluja and Norrbom 1999). Among them, frugivorous flies represent approximately 25–30% of all tephritid species, occur in tropical and temperate regions of all continents except the Antarctic and are predominantly distributed in five main genera (Anastrepha Schiner, Rhagoletis Loew, Ceratitis MacLeay, Dacus Fabricius and Bactrocera Macquart). Frugivorous tephritids attack healthy fruit still on the tree. The larvae develop inside the fruit, feed on the plant tissues, and complete their developmental cycle in the soil. A relatively limited number (approximately 100) of frugivorous species are phytophagous pests whose larvae attack pulp and/or seeds of cultivated fruits and crops of agricultural importance. In Africa, damage on commercial fruits and crops is caused mainly by polyphagous species belonging to the genera Ceratitis, Dacus and Bactrocera (De Meyer et al. 2008; ZooKeys 428: 97–108 (2014) doi: 10.3897/zookeys.428.7366 www.zookeys.org

The fabrication of substrates for surface enhanced Raman spectroscopy (SERS) by printing plasmonic structures on paper has emerged as a potential low-cost replacement for conventional nanofabricated SERS devices. Not only are the paper devices low in cost to produce, the inherent flexibility and fluidic transport capabilities of paper provide advantages in sample collection and processing, as well as analyte concentration. In this study, we review the recent progress in paper or membrane SERS devices fabricated through inkjet printing and other deposition methods. We then report a new potential diagnostics application for paper SERS devices that leverages the fluidic transport and chromatographic capabilities of paper to enable SERS-based detection following PCR. The use of paper SERS creates the potential for a simple yet densely multiplexed PCR assay that is not possible with conventional fluorescence-based transduction.

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.

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.

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.

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.

In this paper, we present an optofluidic SERS microsystem suitable for on-site detection of multiplexed analytics in the field. We show simultaneous detection of three fungicides that are highly regulated in aquaculture. The optofluidic SERS microsystem shows improved portability since it does not require a bulky pump for sample loading. The sample is simply drawn into the microchannel using a pipette. Additionally, two fiber optic cables are inserted into the device for sample excitation and photon collection. The fiber optic cables, aligned into the detection zone, eliminate the need for microscope alignment required in traditional SERS detection. The detection zone of the device consists of a porous matrix of packed silica microspheres that traps silver nanoparticles and adsorbed analyte molecules. The sample is passively concentrated into the matrix as it is loaded by applying negative pressure from the outlet with a pipette. The concentrating matrix has been shown to amplify 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.