Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration.

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses.

Centrifugation-Assisted Three-Dimensional Printing of Devices Embedded with Fully Enclosed Microchannels.

The challenges in reliably removing the sacrificial material from fully enclosed microfluidic channels hinder the use of three-dimensional (3D) printing to create microfluidic devices with intricate geometries. With advances in printer resolution, the etching of sacrificial materials from increasingly smaller channels is poised to be a bottleneck using the existing techniques. In this study, we introduce a microfabrication approach that utilizes centrifugation to effortlessly and efficiently remove the sacrificial materials from 3D-printed microfluidic devices with densely packed microfeatures. We characterize the process by measuring the etch rate under different centrifugal forces and developed a theoretical model to estimate process parameters for a given geometry. The effect of the device layout on the centrifugal etching process is also investigated. We demonstrate the applicability of our approach on devices fabricated using inkjet 3D printing and stereolithography. Finally, the advantages of the introduced approach over commonly used injection-based etching of sacrificial material are experimentally demonstrated in direct comparisons. A robust method to postprocess additively manufactured geometries composed of intricate microfluidic channels can help utilize both the large printing volume and high spatial resolution afforded by 3D printing in creating a variety of devices ranging from scaffolds to large-scale microfluidic assays.

Chemically Amplified Multiplex Detection of SARS-CoV-2 and Influenza A and B Viruses via Paint-Programmed Lateral Flow Assays.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) continues to threaten lives by evolving into new variants with greater transmissibility. Although lateral flow assays (LFAs) are widely used to self-test for coronavirus disease 2019 (COVID-19), these tests suffer from low sensitivity leading to a high rate of false negative results. In this work, a multiplexed lateral flow assay is reported for the detection of SARS-CoV-2 and influenza A and B viruses in human saliva with a built-in chemical amplification of the colorimetric signal for enhanced sensitivity. To automate the amplification process, the paper-based device is integrated with an imprinted flow controller, which coordinates the routing of different reagents and ensures their sequential and timely delivery to run an optimal amplification reaction. Using the assay, SARS-CoV-2 and influenza A and B viruses can be detected with ≈25x higher sensitivity than commercial LFAs, and the device can detect SARS-CoV-2-positive patient saliva samples missed by commercial LFAs. The technology provides an effective and practical solution to enhance the performance of conventional LFAs and will enable sensitive self-testing to prevent virus transmission and future outbreaks of new variants.

Paper-based multi-well depletion ELISA.

Enzyme-linked immunosorbent assay (ELISA) is widely employed for detecting target molecules in bioassays including the serological assays that measure specific antibody titers. However, ELISA tests are inherently limited to centralized laboratories staffed with trained personnel as the assay workflow requires multiple steps to be performed in a specific sequence. Here, we report a dipstick ELISA test that automates this otherwise laborious process and reports the titer of a target molecule in a digital manner without the need for an external instrument or operator. Our assay measures titer by gradually immuno-depleting the target analyte from a flowing sample effectively diluting the residual target - a process conventionally achieved through serially diluting the whole sample in numerous, time-consuming pipetting steps performed manually. Furthermore, the execution of the depletion ELISA process is automated by a built-in flow controller which sequentially delivers different reagents with preset delays. We apply the technology to develop assays measuring (1) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody titers (IgM/IgG antibodies to nucleocapsid and spike protein) and (2) troponin I, a cardiac biomarker.

High throughput, label-free isolation of circulating tumor cell clusters in meshed microwells.

Extremely rare circulating tumor cell (CTC) clusters are both increasingly appreciated as highly metastatic precursors and virtually unexplored. Technologies are primarily designed to detect single CTCs and often fail to account for the fragility of clusters or to leverage cluster-specific markers for higher sensitivity. Meanwhile, the few technologies targeting CTC clusters lack scalability. Here, we introduce the Cluster-Wells, which combines the speed and practicality of membrane filtration with the sensitive and deterministic screening afforded by microfluidic chips. The >100,000 microwells in the Cluster-Wells physically arrest CTC clusters in unprocessed whole blood, gently isolating virtually all clusters at a throughput of >25 mL/h, and allow viable clusters to be retrieved from the device. Using the Cluster-Wells, we isolated CTC clusters ranging from 2 to 100+ cells from prostate and ovarian cancer patients and analyzed a subset using RNA sequencing. Routine isolation of CTC clusters will democratize research on their utility in managing cancer.

Capillary flow control in lateral flow assays via delaminating timers.

Lateral flow assays (LFAs) use capillary flow of liquids for simple detection of analytes. While useful for spontaneously wicking samples, the capillary flow inherently limits performing complex reactions that require timely application of multiple solutions. Here, we introduce a technique to control capillary flow on paper by imprinting roadblocks on the flow path with water-insoluble ink and using the gradual formation of a void between a wetted paper and a sheath polymer tape to create timers. Timers are drawn at strategic nodes to hold the capillary flow for a desired period and thereby enable multiple liquids to be introduced into multistep chemical reactions following a programmed sequence. Using our technique, we developed (i) an LFA with built-in signal amplification to detect human chorionic gonadotropin with an order of magnitude higher sensitivity than the conventional assay and (ii) a device to extract DNA from bodily fluids without relying on laboratory instruments.

Point-of-Care Toolkit for Multiplex Molecular Diagnosis of SARS-CoV-2 and Influenza A and B Viruses.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is still spreading around the globe causing immense public health and socioeconomic problems. As the infection can progress with mild symptoms that can be misinterpreted as the flu, self-testing methods that can positively identify SARS-CoV-2 are needed to effectively track and prevent the transmission of the virus. In this work, we report a point-of-care toolkit for multiplex molecular diagnosis of SARS-CoV-2 and influenza A and B viruses in saliva samples. Our assay is physically programmed to run a sequence of chemical reactions on a paper substrate and internally generate heat to drive these reactions for an autonomous extraction, purification, and amplification of the viral RNA. Using our assay, we could reliably detect SARS-CoV-2 and influenza viruses at concentrations as low as 50 copies/µL visually from a colorimetric analysis. The capability to autonomously perform a traditionally labor-intensive genetic assay on a disposable platform will enable frequent, on-demand self-testing, a critical need to track and contain this and future outbreaks.

Origami paper-based sample preconcentration using sequentially driven ion concentration polarization.

Ion concentration polarization (ICP) is one of the preconcentration techniques which can acquire a high preconcentration factor. Still, the main hurdles of ICP are its instability and low efficiency under physiological conditions with high ionic strength and abundant biomolecules. Here, we suggested a sequentially driven ICP process, which enhanced the electrokinetic force required for preconcentration, enabling enrichment of highly ionic raw samples without increasing the electric field. We acquired a 13-fold preconcentration factor (PF) in human serum using a paper-based origami structure consisting of multiple layers for three-dimensional sequential ICP (3D seq-ICP). Moreover, we demonstrated a paper-based enzyme-linked immunosorbent assay (ELISA) by 3D seq-ICP using tau protein, showing a 6-fold increase in ELISA signals.

Highly efficient and scalable biomarker preconcentrator based on nanoelectrokinetics.

Micro/nanofluidics are excellent candidates for biological sample preparation. However, the limited process volume in micro/nanofluidics is the main hurdle limiting their practical applications. To date, most micro/nanofluidics have processed sample volumes of several microliters and have rarely been used to handle large-volume samples. Herein, we propose a microfluidic paper-based large-volume preconcentrator (u-LVP) for enrichment and purification of biomarkers (e.g., miRNA) using ion concentration polarization. A Nafion (ion-selective nanoporous membrane)-functionalized multilayer cellulose paper enables microscale division of milliliter-scale samples, thus electrokinetically separating and preconcentrating the biomarker in different locations within the u-LVP. By inserting collecting discs at optimal positions in the u-LVP, the enriched biomarker is simply recovered with high efficiency. With this approach, as an exemplary biomarker, miRNA-21 in human serum was separated from proteins and preconcentrated with an effective preconcentration factor exceeding 6.63 and a recovery rate above 84%. Thus, our platform offers new opportunities and benefits for biomarker, diagnostic, prognostic, and therapeutic research.

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes.

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps  of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Combinatorial Immunophenotyping of Cell Populations with an Electronic Antibody Microarray.

Immunophenotyping is widely used to characterize cell populations in basic research and to diagnose diseases from surface biomarkers in the clinic. This process usually requires complex instruments such as flow cytometers or fluorescence microscopes, which are typically housed in centralized laboratories. Microfluidics are combined with an integrated electrical sensor network to create an antibody microarray for label-free cell immunophenotyping against multiple antigens. The device works by fractionating the sample via capturing target subpopulations in an array of microfluidic chambers functionalized against different antigens and by electrically quantifying the cell capture statistics through a network of code-multiplexed electrical sensors. Through a combinatorial arrangement of antibody sequences along different microfluidic paths, the device can measure the prevalence of different cell subpopulations in a sample from computational analysis of the electrical output signal. The device performance is characterized by analyzing heterogeneous samples of mixed tumor cell populations and then the technique is applied to determine leukocyte subpopulations in blood samples and the results are validated against complete blood cell count and flow cytometry results. Label-free immunophenotyping of cell populations against multiple targets on a disposable electronic chip presents opportunities in global health and telemedicine applications for cell-based diagnostics and health monitoring.

Origami-paper-based device for microvesicle/exosome preconcentration and isolation.

Microvesicles and exosomes are promising liquid biopsy biomarkers. However, conventional isolation techniques damage and contaminate the biomarkers. We developed an origami-paper-based device for effective isolation of biomarkers with less damage and in fewer steps. The multi-folded device enables the preconcentration of the microvesicles/exosomes on specific layers (∼5-fold) by the ion concentration polarization technique and they were simply isolated from the rest of the sample by unfolding the device.

Electrokinetic Size-Based Spatial Separation of Micro/Nanospheres Using Paper-Based 3D Origami Preconcentrator.

Sample preparation steps (e.g., preconcentration and separation) are key to enhancing sensitivity and reliability in biomedical and analytical chemistry. However, conventional methods (e.g., ultracentrifugation) cause significant loss of sample as well as their contamination. In this study, we developed a paper-based three-dimensional (3D) origami ion concentration polarization preconcentrator (POP) for highly efficient and facile sample preparation. The unique design of POP enables simultaneous preconcentration and spatial separation of target analytes rapidly and economically. The POP comprises accordion-like multifolded layers with convergent wicking areas that can separate analytes based on their sizes in different layers, which can then be easily isolated by unfolding the POP. We first demonstrated 100-fold preconcentration of albumin and its isolation on the specific layers. Then, we demonstrated the simultaneous preconcentration and spatial separation of microspheres of three different sizes (with diameters of 0.02, 0.2, and 2 µm) on the different layers.

Battery operated preconcentration-assisted lateral flow assay.

Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial ß-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 µW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

Low-cost and facile fabrication of a paper-based capillary electrophoresis microdevice for pathogen detection.

This paper describes the development of a novel paper-based capillary electrophoresis (pCE) microdevice using mineral paper, which is durable, oil and tear resistant, and waterproof. The pCE device is inexpensive (~$1.6 per device for materials), simple to fabricate, lightweight, and disposable, so it is more adequate for point-of-care (POC) pathogen diagnostics than a conventional CE device made of glass, quartz, silicon or polymer. In addition, the entire fabrication process can be completed within 1h without using expensive clean room facilities and cumbersome photolithography procedures. A simple cross-designed pCE device was patterned on the mineral paper by using a plotter, and assembled with an OHP film via a double-sided adhesive film. After filling the microchannel with polyacrylamide gel, the injection, backbiasing, and separation steps were sequentially operated to differentiate single-stranded DNA (ssDNA) with 4 bp resolution in a 2.9cm-long CE separation channel. Furthermore, we successfully demonstrated the identification of the PCR amplicons of two target genes of Escherichia coli O157:H7 (rrsH gene, 121 bp) and Staphylococcus aureus (glnA gene, 225 bp). For accurate assignment of the peaks in the electropherogram, two bracket ladders (80 bp for the shortest and 326 bp for the longest) were employed, so the two amplicons of the pathogens were precisely identified on a pCE chip within 3min using the relative migration time ratio without effect of the CE environments. Thus, we believe that the pCE microdevice could be very useful for the separation of nucleic acids, amino acids, and ions as an analytical tool for use in the medical applications in the resource-limited environments as well as fundamental research fields.

Simple and Highly Sensitive Molecular Diagnosis of Zika Virus by Lateral Flow Assays.

We have developed a simple, user-friendly, and highly sensitive Zika virus (ZIKV) detection method by incorporating optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a lateral flow assay (LFA). The optimized RT-LAMP reaction was carried out using Bst 3.0 polymerase, which has robust and fast isothermal amplification performance even in the presence of high concentrations of inhibitors; this permitted the amplification of ZIKV RNA in pure water and human whole blood. In addition, the strong reverse transcription activity of Bst 3.0 polymerase enabled specific ZIKV RNA amplification without extra addition of reverse transcriptase. The RT-LAMP condition was optimized by adjusting the Mg2+ and dNTP mix concentration to extirpate nontarget amplification, which is caused by nonspecific primer dimers amplification. After 30 min of RT-LAMP reaction, the resultant amplicons were simply and rapidly analyzed by the LFA test in less than 5 min. The optimized RT-LAMP combined with the LFA allowed specific ZIKV RNA detection down to the single copy level within 35 min.

Total integrated slidable and valveless solid phase extraction-polymerase chain reaction-capillary electrophoresis microdevice for mini Y chromosome short tandem repeat genotyping.

A fully integrated slidable and valveless microsystem, which performs solid phase DNA extraction (SPE), micro-polymerase chain reaction (µPCR) and micro-capillary electrophoresis (µCE) coupled with a portable genetic analyser, has been developed for forensic genotyping. The use of a slidable chip, in which a 1 µL-volume of the PCR chamber was patterned at the center, does not necessitate any microvalves and tubing systems for fluidic control. The functional micro-units of SPE, µPCR, and µCE were fabricated on a single glass wafer by conventional photolithography, and the integrated microdevice consists of three layers: from top to bottom, a slidable chip, a channel wafer in which a SPE chamber, a mixing microchannel, and a CE microchannel were fabricated, and a Ti/Pt resistance temperature detector (RTD) wafer. The channel glass wafer and the RTD glass wafer were thermally bonded, and the slidable chip was placed on the designated functional unit. The entire process from the DNA extraction using whole human blood sample to identification of target Y chromosomal short tandem repeat (STR) loci was serially carried out with simply sliding the slidable chamber from one to another functional unit. Monoplex and multiplex detection of amelogenin and mini Y STR loci were successfully analysed on the integrated slidable SPE-µPCR-µCE microdevice by using 1 µL whole human blood within 60 min. The proposed advanced genetic analysis microsystem is capable of point-of-care DNA testing with sample-in-answer-out capability, more importantly, without use of complicated microvalves and microtubing systems for liquid transfer.

An integrated direct loop-mediated isothermal amplification microdevice incorporated with an immunochromatographic strip for bacteria detection in human whole blood and milk without a sample preparation step.

We have developed an integrated direct loop-mediated isothermal amplification (Direct LAMP) microdevice incorporated with an immunochromatographic strip (ICS) to identify bacteria contaminated in real samples. The Direct LAMP is a novel isothermal DNA amplification technique which does not require thermal cycling steps as well as any sample preparation steps such as cell lysis and DNA extraction for amplifying specific target genes. In addition, the resultant amplicons were colorimetrically detected on the ICS, thereby enabling the entire genetic analysis process to be simplified. The two functional units (Direct LAMP and ICS) were integrated on a single device without use of the tedious and complicated microvalve and tubing systems. The utilization of a slidable plate allows us to manipulate the fluidic control in the microchannels manually and the sequential operation of the Direct LAMP and ICS detection could be performed by switching the slidable plate to each functional unit. Thus, the combination of the direct isothermal amplification without any sample preparation and thermal cycling steps, the ICS based amplicon detection by naked eyes, and the slidable plate to eliminate the microvalves in the integrated microdevice would be an ideal platform for point-of-care DNA diaganotics. On the integrated Direct LAMP-ICS microdevice, we could analyze Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) contaminated in human whole blood or milk at a single-cell level within 1h.

Correction: An integrated slidable and valveless microdevice with solid phase extraction, polymerase chain reaction, and immunochromatographic strip parts for multiplex colorimetric pathogen detection.

Correction for 'An integrated slidable and valveless microdevice with solid phase extraction, polymerase chain reaction, and immunochromatographic strip parts for multiplex colorimetric pathogen detection' by Yong Tae Kim et al., Lab Chip, 2015, 15, 4148-4155.

An integrated slidable and valveless microdevice with solid phase extraction, polymerase chain reaction, and immunochromatographic strip parts for multiplex colorimetric pathogen detection.

A total integrated genetic analysis microsystem was developed, which consisted of solid phase extraction (SPE), polymerase chain reaction (PCR), and immunochromatographic strip (ICS) parts for multiplex colorimetric detection of pathogenic Staphylococcus aureus (S. aureus) and Escherichia coli O157:H7 (E. coli O157:H7) on a portable genetic analyzer. Utilizing a slidable chamber, which is a movable glass wafer, complex microvalves could be eliminated for fluidic control in the microchannel, which could simplify the chip design and chip operation. The integrated slidable microdevice was composed of 4 layers: a 4-point Pt/Ti resistance temperature detector (RTD) wafer, a micro-patterned channel wafer, a 2 µL volume slidable chamber, and an ICS. The entire process from the DNA extraction in the SPE chamber to the detection of the target gene expression by the ICS was serially performed by simply sliding the slidable chamber from one part to another functional part. The total process for multiplex pathogenic S. aureus and E. coli O157:H7 detection on the integrated slidable microdevice was accomplished within 55 min with a detection limit of 5 cells. Furthermore, spiked bacteria samples in milk were also successfully analysed on the portable genetic analysis microsystem with sample-in-answer-out capability. The proposed total integrated microsystem is adequate for point-of-care DNA testing in that no microvalves and complex tubing systems are required due to the use of the slidable chamber and the bulky and expensive fluorescence or electrochemical detectors are not necessary due to the ICS based colorimetric detection.