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Organic materials have been widely used in photovoltaic solar applications because of their flexibility and low cost. Poly(3,4-ethylenedioxythiophene) (PEDOT) has been known to have good mechanical stability under stretching and bending and also has good electrical and optical properties. Unlike inorganic materials, PEDOT can withstand high strains. In flexible solar cells, modules are usually exposed to heating–cooling cycles during day and night. When the structure of a thin film on a flexible substrate is exposed to high temperatures, the film and the substrate will expand at different rates; this will produce a residual strain caused by the thermal mismatch between the two layers. In contrast, indium–tin oxide (ITO) is brittle, and its conductivity decreases significantly as the ITO films are exposed to stretching or bending strains, especially in flexible/foldable solar cell applications. Cracks in ITO appear at very low strains that might cause failure in the conductive layer because of the combination of a very thin film of brittle ceramic material applied to a polymer substrate. In this work, a comparison of the thermal stability was conducted between PEDOT and ITO on flexible polyethylene terephthalate (PET) substrate. The performances of the two candidates were assessed during thermal bending fatigue and accelerated thermal cycling experiments. This work was designed to study the behavior of ITO and PEDOT under thermal cycling and thermal bending fatigue.

One of the grand challenges for field-deployable NATs is related to the front end of the assays – nucleic acid extraction from raw samples. The ideal nucleic acid sample preparation should be simple, scalable, and easy-to-operate. In this chapter, we present a lab-on-a-disc NAT device for sample-to-answer malaria diagnosis. The parasite DNA sample preparation and subsequent real-time LAMP detection were seamlessly integrated on a disposable single microfluidic compact disc, driven by energy-efficient, non-centrifuge based magnetic field interactions. Each disc contains four parallel testing units, which could be configured either as four identical tests or as four species-specific tests. When configured as species-specific tests, it could identify two of the most life-threatening malaria species (P. falciparum and P. vivax). The reagent disc with a 4-plex analyzer (discussed in Chapter 1) is capable of processing four samples simultaneously with 40 min turnaround time. It achieves a detection limit of ~0.5 parasites/μl for whole blood, sufficient for detecting asymptomatic parasite carriers. The assay is performed with an automated device described in the companion chapter. The combination of sensitivity, specificity, cost, and scalable sample preparation suggests the real-time fluorescence LAMP device could be particularly useful for malaria screening in the field settings.

Low-cost access to the highly sensitive and specific detection of the pathogen in the field is a crucial attribute for the next generation point-of-care (POC) platforms. In this work, we developed a real-time fluorescence nucleic acid testing device with automated and scalable sample preparation capability for field malaria diagnosis. The palm-sized battery-powered analyzer equipped with a disposable microfluidic reagent compact disc described in the companion Chap. 16 which facilitates four isothermal nucleic acid tests in parallel from raw blood samples to answer. The platform has a user-friendly interface such as touchscreen LCD and smartphone data connectivity for on-site and remote healthcare delivery, respectively. The chapter mainly focuses on describing integration procedures of the real-time fluorescence LAMP analyzer and the validation of its subsystems. The device cost is significantly reduced compared to the commercial benchtop real-time machine and other existing POC platforms. As a platform technology, self-sustainable, portable, low-cost, and easy-to-use analyzer design should create a new paradigm of molecular diagnosis toward a variety of infectious diseases at the point of need.

HIV self-testing is an emerging innovative approach that allows individuals who want to know their HIV status to collect their own specimen, perform a test, and interpret the results privately. Existing HIV self-testing methods rely on rapid diagnostic tests (RDTs) to detect the presence of HIV-1/2 antibodies, which could miss a significant portion of asymptomatic carriers during the window period. In this work, we present a fully integrated nucleic acid testing (NAT) device towards streamlined HIV self-testing using 100 μL finger-prick whole blood. The device consists of a ready-to-use microfluidic reagent cartridge and an ultra-compact NAT-on-USB analyzer. The test requires simple steps from the user to drop the finger-prick blood sample into a collection tube with lysis buffer and load the lysate onto the microfluidic cartridge, and the testing result can be easily read out by a custom-built graphical user interface (GUI). The microfluidic cartridge and the analyzer automatically handle the complexity of sample preparation, purification, and real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP). With a turnaround time of ∼60 min, we achieved a limit of detection (LoD) of 214 viral RNA copies/mL of whole blood at a 95% confidence level. Due to its ease of use and high sensitivity, we anticipate the HIV NAT-on-USB device would be particularly useful for the high-risk populations seeking private self-testing at the early stages of exposure.

Solid-state nanopores have shown great promise and achieved tremendous success in label-free single-molecule analysis. However, there are three common challenges in solid-state nanopore sensors, including the nanopore size variations from batch to batch that makes the interpretation of the sensing results difficult, the incorporation of sensor specificity, and the impractical analysis time at low analyte concentration due to diffusion-limited mass transport. Here, we demonstrate a novel loop-mediated isothermal amplification (LAMP)-coupled glass nanopore counting strategy that could effectively address these challenges. By using the glass nanopore in the counting mode (versus the sizing mode), the device fabrication challenge is considerably eased since it allows a certain degree of pore size variations and no surface functionalization is needed. The specific molecule replication effectively breaks the diffusion-limited mass transport thanks to the exponential growth of the target molecules. We show the LAMP-coupled glass nanopore counting has the potential to be used in a qualitative test as well as in a quantitative nucleic acid test. This approach lends itself to most amplification strategies as long as the target template is specifically replicated in numbers. The highly sensitive and specific sensing strategy would open a new avenue for solid-state nanopore sensors toward a new form of compact, rapid, low-cost nucleic acid testing at the point of care.

Due to its simplicity and robustness, pore-based resistive pulse sensors have been widely used to detect, measure, and analyze particles at length scales ranging from nanometers to micrometers. While multiple pore-based resistive pulse sensors are preferred to increase the analysis throughput and to overcome the clogging issues, the scalability is often limited. In response, by combining the time-division multiple access technique in the telecommunication field with the microfluidics, we reported a microfluidic time-division multiplexing accessing (TDMA) single-end resistive pulse sensor, in which particles can be analyzed through a scalable number of microfluidic channels. With an eight-channel microfluidic device and polystyrene particles as proof-of-principle, we successfully demonstrated this multiplexed technology is effective in measuring the particle size and concentration, in analyzing the particle arriving dynamics, and in discriminating mixed populations. Importantly, the availability of multiple sensing pores provides a robust mechanism to overcome the clogging issue, allowing the analysis to continue even when some of the pores are clogged. We anticipate this TDMA approach could find wide applications and facilitate future development of multiplexed resistive pulse sensing from the microscale to nanoscale.

Introduction: Over the past decade, loop-mediated isothermal amplification (LAMP) technology has played an important role in molecular diagnostics. Amongst numerous nucleic acid amplification assays, LAMP stands out in terms of sample-to-answer time, sensitivity, specificity, cost, robustness, and accessibility, making it ideal for field-deployable diagnostics in resource-limited regions.Areas covered: In this review, we outline the front-end LAMP design practices for point-of-care (POC) applications, including sample handling and various signal readout methodologies. Next, we explore existing LAMP technologies that have been validated with clinical samples in the field. We summarize recent work that utilizes reverse transcription (RT) LAMP to rapidly detect SARS-CoV-2 as an alternative to standard PCR protocols. Finally, we describe challenges in translating LAMP from the benchtop to the field and opportunities for future LAMP assay development and performance reporting.Expert opinion: Despite the popularity of LAMP in the academic research community and a recent surge in interest in LAMP due to the COVID-19 pandemic, there are numerous areas for improvement in the fundamental understanding of LAMP, which are needed to elevate the field of LAMP assay development and characterization.

We demonstrate the feasibility of using the single molecule sensing nanopore as a digital counter to enumerate the amplicons for ultrasensitive electronic nucleic acid analysis. We show that the nanopore digital counting approach could capture the DNA replication dynamics in the LAMP reaction and has the potential to be used in a qualitative test as well as in a quantitative test. By keeping nanopores as simple as possible and by leveraging the amplification assay’s high sensitivity, the amplification-coupled nanopore digital counting approach provides a promising optics-free method for highly sensitive and specific nucleic acid testing.

The mechanical behavior of individual cells plays an important role in regulating various biological activities at the molecular and cellular levels. It can serve as a promising label-free marker of cells’ physiological states. In the past two decades, several techniques have been developed for understanding correlations between cellular mechanical changes and human diseases. However, numerous technical challenges remain with regard to realizing high-throughput, robust, and easy-to-perform measurements of single-cell mechanical properties. In this paper, we review the emerging tools for single-cell mechanical characterization that are provided by microfluidic technology. Different techniques are benchmarked by considering their advantages and limitations. Finally, the potential applications of microfluidic techniques based on cellular mechanical properties are discussed.

Due to its simplicity and robustness, resistive pulse sensors have been widely used to detect, measure, and analyze particles at length scales ranging from nanometers to micrometers. While multiple pore-based resistive pulse sensors are preferred to increase the analysis throughput and to overcome the clogging issues, the scalability is often limited. Here we reported a microfluidic time-division multiplexing accessing (TDMA) single-end resistive pulse sensor, in which particles can be analyzed through a scalable number of microfluidic channels. This multiplexed approach is effective in measuring the particle size and concentration, in analyzing the particle arriving dynamics, and in discriminating mixed populations. It also provides a robust mechanism to overcome the clogging issue, allowing the analysis to continue even when some of the pores are clogged.

Most lab-on-a-disc platforms use centrifugal force to pump liquids. In this work, we demonstrated a novel energy efficient non-centrifugal lab-on-a-disc nucleic acid testing device in which no liquid motion is involved. Streamlined DNA extraction, amplification, and real-time detection on a single microfluidic reagent disc are achieved by actuating the DNA-carrying magnetic beads against stationary reagent droplets. Using malaria spiked whole blood as a testing model, we demonstrated an excellent detection limit of 0.5 parasites/µl, sufficient for detecting asymptomatic malaria parasite carriers.

Mechanical properties have emerged as a significant label-free marker for characterizing deformable particles such as cells. Here, we demonstrated the first single-particle-resolved, cytometry-like deformability-activated sorting in the continuous flow on a microfluidic chip. Compared with existing deformability-based sorting techniques, the microfluidic device presented in this work measures the deformability and immediately sorts the particles one-by-one in real time. It integrates the transit-time-based deformability measurement and active hydrodynamic sorting onto a single chip. We identified the critical factors that affect the sorting dynamics by modeling and experimental approaches. We found that the device throughput is determined by the summation of the sensing, buffering, and sorting time. A total time of ~100 ms is used for analyzing and sorting a single particle, leading to a throughput of 600 particles/min. We synthesized poly(ethylene glycol) diacrylate (PEGDA) hydrogel beads as the deformability model for device validation and performance evaluation. A deformability-activated sorting purity of 88% and an average efficiency of 73% were achieved. We anticipate that the ability to actively measure and sort individual particles one-by-one in a continuous flow would find applications in cell-mechanotyping studies such as correlational studies of the cell mechanical phenotype and molecular mechanism. Researchers in the United States have developed a microfluidic device that sorts particles in real-time based on their deformability. The deformability of cells changes in diseases such as cancer and malaria, as well as disorders like sickle cell anemia, making it an attractive biomarker. A team at Pennsylvania State University led by Weihua Guan engineered a device to sort mixed samples based on real-time deformability measurements. The device determines deformability based on how long a particle takes to squeeze through a sensing pore. A pair of valves then directs the particle to the appropriate outlet through pneumatic control. A test sorting of hydrogel beads revealed a throughput of 600 particles/min with a sorting efficiency of 73% and an 88% accuracy. The device will be useful for researchers investigating the mechanical properties and dynamics of cells.

The effectiveness of malaria screening and treatment highly depends on the low-cost access to the highly sensitive and specific malaria test. We report a real-time fluorescence nucleic acid testing device for malaria field detection with automated and scalable sample preparation capability. The device consists a compact analyzer and a disposable microfluidic reagent compact disc. The parasite DNA sample preparation and subsequent real-time LAMP detection were seamlessly integrated on a single microfluidic compact disc, driven by energy efficient non-centrifuge based magnetic field interactions. Each disc contains four parallel testing units which could be configured either as four identical tests or as four species-specific tests. When configured as species-specific tests, it could identify two of the most life-threatening malaria species (P. falciparum and P. vivax). The NAT device is capable of processing four samples simultaneously within 50 min turnaround time. It achieves a detection limit of ~0.5 parasites/µl for whole blood, sufficient for detecting asymptomatic parasite carriers. The combination of the sensitivity, specificity, cost, and scalable sample preparation suggests the real-time fluorescence LAMP device could be particularly useful for malaria screening in the field settings.

In order to improve the performance of various flexible electronic devices, the research on the transparent conductive thin films becomes very intensive in the recent years. In this work, we studied mechanical and thermal behaviors of two types of transparent conductive thin films, Poly(3,4-ethylenedioxythiophene) (PEDOT) as an example on polymer conductive thin film, and indium tin oxide (ITO) as an example on transparent conductive oxide thin film. Both films are deposited on polyethyleneterephthalate (PET) substrate. Two sheet resistances for PEDOT (i.e., 150 Ω/sq and 225 Ω/sq) and one sheet resistance for ITO (i.e., 60 Ω/sq) were involved in the study. PEDOT showed good mechanical properties with a small electrical resistance change and no clear cracks or deformation on the film surface under the condition of cyclic bending. However, the resistance of ITO significantly increased with the cyclic bending and cracks were seen initiated in the center of sample and propagated toward the edges. Further, design of experiment approach was used to study the effect of different cyclic bending parameters, such as bending diameter and frequency. Additionally, damp heat experiment on similar samples was conducted by applying 85 °C temperatures and 85% relative humidity on them for 1000 h. The electrical resistance was dramatically increased for both films. Scanning electronic microscopy (SIM), Energy Dispersive Spectrometry (EDX) and transmission tests were also used to determine the change of films’ compositions and transparencies.

The goal of this work is to pursue analytical approaches that elucidate electron and proton diffusion inside the Shewanella oneidensis biofilm and bulk liquid, which will inevitably promote the translation of Microbial Fuel Cell (MFC) technology for renewable, "green energy" solutions that are in demand to sustain the world's ever-increasing energy demands and to mitigate the depletion of current resources. This study provides a novel strategy for monitoring electron/proton fluxes in 3-D multi-laminate structures of paper as a scaffold to support S. oneidensis biofilms and bulk media liquid. Multiple layers of paper containing bacterial cells and/or media are stacked to form a layered 3-D model of the overall biofilm/bulk liquid construct. Mass transport of electrons and protons into this 3-D system can be quantified along with the exploration of microbial energy production. Assembly of a 3D paper stack can be modular and allows us to control the thickness of the overall biofilm/bulk liquid construct with the different diffusion distances of the electrons/protons through the stack. By measuring the current generated from the 3-D stack, the electron and proton diffusivity through biofilms were quantitatively investigated. We found that (i) the diffusion length of the electrons/protons in the S. oneidensis biofilm/bulk liquid is a determinant factor for the MFC performance, (ii) the electron transfer through the endogenous mediators of S. oneidensis can be a more critical factor to limit the current/power generation of the MFCs than the proton transfer in the MFC system and (iii) the thicker biofilm allows higher and longer current generation but requires more time to reach a peak current value and increases the total energy loss of the MFC system.

This work reports a high throughput and label-free cell deformability microfluidic sensor for quantitative parasitemia measurement and stage determination for Plasmodium falciparum-infected red blood cells (Pf-iRBCs). As a mechanical biomarker, the RBC deformability is highly relevant to the infection status. The cell deformability is measured by evaluating the translocation time when each individual cell squeezes through a microscale constriction. More than 30,000 RBCs can be analyzed for parasitemia quantification in under 1 min with a throughput ∼500 cells/s. Moreover, the device can also differentiate various malaria stages (ring, trophozoite, and schizont stage) due to their varied deformability. As compared to the microscopy and flow cytometry, this microfluidic deformability sensor would allow for label-free and rapid malaria parasitemia quantification and stage determination at a low-cost.

In response to the urgent need of a field-deployable and highly sensitive malaria diagnosis, we developed a standalone, "sample-in-answer-out" molecular diagnostic system (AnyMDx) to enable quantitative molecular analysis of blood-borne malaria in low resource areas. The system consists of a durable battery-powered analyzer and a disposable microfluidic compact disc loaded with reagents ready for use. A low power thermal module and a novel fluorescence-sensing module are integrated into the analyzer for real-time monitoring of loop-mediated isothermal nucleic acid amplification (LAMP) of target parasite DNA. With 10 μL of raw blood sample, the AnyMDx system automates the nucleic acid sample preparation and subsequent LAMP and real-time detection. Under laboratory conditions with whole-blood samples spiked with cultured Plasmodium falciparum, we achieved a detection limit of ∼0.6 parasite per μL, much lower than those for the conventional microscopy and rapid diagnostic tests (∼50-100 parasites per μL). The turnaround time from sample to answer is less than 40 minutes. The AnyMDx is user-friendly requiring minimal technological training. The analyzer and the disposable reagent compact discs are cost-effective, making AnyMDx a potential tool for malaria molecular diagnosis under field settings for malaria elimination.

This paper presents a "sample-in-answer-out" molecular diagnostic system (AnyMDx) for blood-born malaria detection at the point of care, especially in resource-limiting settings. AnyMDx consists of a microfluidic reagent compact disk incorporating seamlessly integrated steps of DNA extraction, purification, elution, and amplification, as well as a mobile battery-powered analyzer. Low power thermal module and novel fluorescence sensing module are integrated into the analyzer for real-time monitoring of loop-mediated isothermal nucleic acid amplification (LAMP). The AnyMDx has an assay detection limit of ∼0.6 parasites/μL against blood P. falciparum parasites, much lower than conventional microscope-based method (∼50 parasites/μL) and immunoassay based RTD (∼100 parasites/μL). AnyMDx is fully automated and thus is easily accessible to non-specialist with minimal molecular training. The sample-to-answer turnaround time for AnyMDx is less than 40 minutes.

Coulter counters (a.k.a. resistive pulse sensors) were widely used to measure the size of biological cells and colloidal particles. One of the important parameters of Coulter counters is its size discriminative capability. This work reports a multiple pore-based microfluidic Coulter counter for improved size differentiation in a mixed sample. When a single particle translocated across an array of sensing pores, multiple time-related resistive pulse signals were generated. Due to the time correlation of these resistive pulse signals, we found a multiple cross-correlation analysis (MCCA) could enhance the sizing signal-to-noise (SNR) ratio by a factor of n1/2, where n is the pore numbers in series. This proof-of-concept is experimentally validated with polystyrene beads as well as human red blood cells. We anticipate this method would be highly beneficial for applications where improved size differentiation is required.

Indium Tin Oxide (ITO) has been widely used as a Transparent Conductive Oxide (TCO) layer in the photovoltaic solar technology because of its excellent electrical and optical properties. However, ITO is brittle, and its conductivity decreases significantly as the ITO films are exposed to stretching or bending strains especially in flexible/foldable solar cell applications. The cracks in ITO appear at very low strains which might cause failure in the conductive layer because of the combination of a very thin film of brittle ceramic material applied to a polymer substrate. Poly (3, 4-ethylenedioxythiophene), abbreviated PEDOT, is of increasing interest as a competitive candidate to ITO. PEDOT has found its way in many applications such as transparent electrode materials and transparent conductive layers in photovoltaic solar cells. In this work, the mechanical behavior of PEDOT was studied under high cycle bending fatigue in which the effects of bending diameter and bending frequency were considered and compared to ITO. High magnification optical images were used to study cracking in the PEDOT as well as the ITO layers. In flexible solar cells, the web will be exposed to folding/bending many times during manufacturing and installation. Therefore, the thin film substrate structure will be exposed to cyclic loading cyclic tensile and compressive strains. Therefore, this work was designed to mechanically fatigue the structure and study its behavior. It was found that bending diameters as well as material (PEDOT or ITO) have a great influence on the electrical conductivity of the thin films.

Microfluidics-based drug-screening systems have enabled efficient and high-throughput drug screening, but their routine uses in ordinary labs are limited due to the complexity involved in device fabrication and system setup. In this work, we report an easy-to-use and low-cost arbitrarily accessible 3D microfluidic device that can be easily adopted by various labs to perform combinatorial assays for high-throughput drug screening. The device is capable of precisely performing automatic and simultaneous reagent loading and aliquoting tasks and performing multistep assays with arbitrary sequences. The device is not intended to compete with other microfluidic technologies regarding ultra-low reaction volume. Instead, its freedom from tubing or pumping systems and easy operation makes it an ideal platform for routine high-throughput drug screening outside traditional microfluidic labs. The functionality and quantitative reliability of the 3D microfluidic device were demonstrated with a histone acetyltransferase-based drug-screening assay using the recombinant Plasmodium falciparum GCN5 enzyme, benchmarked with a traditional microtiter plate-based method. This arbitrarily accessible, multistep capable, low-cost, and easy-to-use device can be widely adopted in various combinatorial assays beyond high-throughput drug screening.

In this work, we provided a strategy for high-throughput monitoring of temporal and spatial gradients of electrons/protons in electrogenic bacterial biofilms by utilizing 3D multi-laminate structures of paper as a scaffold to support bacterial biofilms and/or media. Assembly of a 3D paper stack can be modular and allows us to control the thickness of the overall biofilm construct and diffusion of nutrients/redox mediators through the stack and the shapes of gradients in the stacks can be modulated by changing the composition of each layer. By measuring the current generated from different stack configurations, the electron/proton fluxes through biofilms were quantitatively investigated.

In this work, we provided a new technique to monitor the flow of bacterial cells in paper. This was based on real-time measuring of the electricity generated from bacterial metabolism. Despite recent efforts to monitor liquid flow in paper, there is not yet a technique that provides detailed flow information of particles in paper microfluidics. Our device contained three hydrophilic spots linked by a hydrophilic channel on paper. Each spot came into contact with an anode that was electrically connected to a cathode through an external load. When bacterial cells were transported to each spot by capillary force, the current generated from the bacteria was monitored and bacterial cells' transportation through the channel was quantitatively investigated according to the pore size of the paper.

We demonstrate the use of a paper-based sensing platform for rapid and high-throughput characterization of microbial electricity-generating capabilities. For the first time, a 48-well microbial fuel cell (MFC) array was fabricated on paper substrates, providing 48 high-throughput measurements and highly comparable performance characteristics in a reliable manner. Spatially distinct 48 wells of the sensor array were prepared by patterning 48 hydrophilic reservoirs in paper with hydrophobic wax boundaries. The paper-based platform exploited the ability of paper to quickly wick fluid and promote bacterial attachment to the gold anode pads, resulting in an instant current generation upon loading of bacterial inoculum and catholyte. This paper-based 48-well MFC array does not require external pumps/tubings and represents the most rapid and the highest throughput test platform for electrogenic bacterial screening.