Cancer Methylation Biomarker Detection in an Automated, Portable, Multichannel Magnetofluidic Platform.

Early detection of cancer is critical to improving clinical outcomes, especially in territories with limited healthcare resources. DNA methylation biomarkers have shown promise in early cancer detection, but typical workflows require highly trained personnel and specialized equipment for manual and lengthy processing, limiting use in resource-constrained areas. As a potential solution, we introduce the Automated Cartridge-based Cancer Early Screening System (ACCESS), a compact, portable, multiplexed, automated platform that performs droplet magnetofluidic- and methylation-specific qPCR-based assays for the detection of DNA methylation cancer biomarkers. Development of ACCESS focuses on esophageal cancer, which is among the most prevalent cancers in low- and middle-income countries with extremely low survival rates. Upon implementing detection assays for two esophageal cancer methylation biomarkers within ACCESS, we demonstrated successful detection of both biomarkers from esophageal tumor tissue samples from eight esophageal cancer patients while showing specificity in paired normal esophageal tissue samples. These results illustrate ACCESS's potential as an amenable epigenetic diagnostic tool for resource-constrained areas toward early detection of esophageal cancer and potentially other malignancies.

Streamlined instrument-free lysis for the detection of Candida auris.

The continued spread of Candida auris in healthcare facilities has increased the demand for widely available screening to aid in containment and inform treatment options. Current methods of detection can be unreliable and require bulky and expensive instruments to lyse and identify fungal pathogens. Here, we present a quick, low-cost, instrument-free method for lysis of C. auris suitable for streamlined sample processing with polymerase chain reaction (PCR) detection. Chemical, thermal, and bead beating lysis techniques were evaluated for lysis performance and compatibility with nucleic acid extraction and downstream PCR reactions. Using only 10 s of manual shaking with glass beads, this method demonstrated a limit of detection (LOD) of C. auris at 500 colony forming units per mL, a 20-fold improvement compared to the LOD without manual shaking, and a 60-fold reduction in time compared to common fungal lysis kits, all while maintaining repeatability and reproducibility across multiple users. This work highlights a simple method for increasing sensitivity and reducing turnaround time of PCR-based C. auris detection and exhibits promise for integration into point-of-care platforms towards real-time triage of colonized patients.

Rapid and Portable Quantification of HIV RNA via a Smartphone-enabled Digital CRISPR Device and Deep Learning.

For the 28.2 million people in the world living with HIV/AIDS and receiving antiretroviral therapy, it is crucial to monitor their HIV viral loads with ease. To this end, rapid and portable diagnostic tools that can quantify HIV RNA are critically needed. We report herein a rapid and quantitative digital CRISPR-assisted HIV RNA detection assay that has been implemented within a portable smartphone-based device as a potential solution. Specifically, we first developed a fluorescence-based reverse transcription recombinase polymerase amplification (RT-RPA)-CRISPR assay for isothermally and rapidly detecting HIV RNA at 42 °C in < 30 min. When realized within a commercial stamp-sized digital chip, this assay yields strongly fluorescent digital reaction wells corresponding to HIV RNA. The isothermal reaction condition and the strong fluorescence in the small digital chip unlock compact thermal and optical components in our device, allowing us to engineer a palm-size (70 × 115 × 80 mm) and lightweight (< 0.6 kg) device. Further leveraging the smartphone, we wrote a custom app to control the device, perform the digital assay, and acquire fluorescence images throughout the assay time. We additionally trained and verified a Deep Learning-based algorithm for analyzing fluorescence images and detecting strongly fluorescent digital reaction wells. Using our smartphone-enabled digital CRISPR device, we were able to detect 75 copies of HIV RNA in 15 min and demonstrate the potential of our device toward convenient monitoring of HIV viral loads and combating the HIV/AIDS epidemic.

Point-of-Care Amenable Detection of Mycoplasma genitalium and Its Antibiotic Resistance Mutations.

Mycoplasma genitalium (MG) is an emerging sexually transmitted bacterium. Due to its fastidious and slow-growing nature, MG is difficult to detect through culture-based diagnostics. Like Neisseria gonorrheae, another bacterial pathogen linked to sexually transmitted infections (STIs), MG has developed resistance to macrolide and fluoroquinolone antibiotics used to treat STIs. The ability to detect MG and identify genomic mutations associated with antibiotic resistance simultaneously can enable antibiotic stewardship and mitigate the spread of antibiotic-resistant MG. Toward this end, we first developed a multiplexed probe-based PCR-melt assay that detects MG and the presence of macrolide resistance mutations in the 23S rRNA gene and fluoroquinolone resistance mutations in the parC gene. Each target was identified via its unique combination of fluorescence label and melting temperature. This approach allowed differentiation between the different types of mutations at the genes of interest. Following initial assay optimization, the assay was integrated into a droplet magnetofluidic cartridge used in a portable platform to integrate automated sample extraction, PCR amplification, and detection. Lastly, we demonstrated that the integrated assay and droplet magnetofluidic platform could detect MG and antibiotic resistance-associated mutations in clinical isolates spiked into urine samples in 40 min.

Sensitive and Quantitative Point-of-Care HIV Viral Load Quantification from Blood Using a Power-Free Plasma Separation and Portable Magnetofluidic Polymerase Chain Reaction Instrument.

Point-of-care (POC) HIV viral load (VL) tests are needed to enhance access to HIV VL testing in low- and middle-income countries (LMICs) and to enable HIV VL self-testing at home, which in turn have the potential to enhance the global management of the disease. While methods based on real-time reverse transcription-polymerase chain reaction (RT-PCR) are highly sensitive and quantitatively accurate, they often require bulky and expensive instruments, making applications at the POC challenging. On the other hand, although methods based on isothermal amplification techniques could be performed using low-cost instruments, they have shown limited quantitative accuracies, i.e., being only semiquantitative. Herein, we present a sensitive and quantitative POC HIV VL quantification method from blood that can be performed using a small power-free three-dimensional-printed plasma separation device and a portable, low-cost magnetofluidic real-time RT-PCR instrument. The plasma separation device, which is composed of a plasma separation membrane and an absorbent material, demonstrated 96% plasma separation efficiency per 100 µL of whole blood. The plasma solution was then processed in a magnetofluidic cartridge for automated HIV RNA extraction and quantification using the portable instrument, which completed 50 cycles of PCR in 15 min. Using the method, we achieved a limit of detection of 500 HIV RNA copies/mL, which is below the World Health Organization's virological failure threshold, and a good quantitative accuracy. The method has the potential for sensitive and quantitative HIV VL testing at the POC and at home self-testing.

Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification.

Polymerase chain reaction (PCR)-based diagnostic testing is the gold standard method for pathogen identification (ID) with recent developments enabling automated PCR tests for point-of-care (POC) use. However, multiplexed identification of several pathogens in PCR assays typically requires optics for an equivalent number of fluorescence channels, increasing instrumentation's complexity and cost. In this study, we first developed ratiometric PCR that surpassed one target per color barrier to allow multiplexed identification while minimizing optical components for affordable POC use. We realized it by amplifying pathogenic targets with fluorescently labeled hydrolysis probes with a specific ratio of red-to-green fluorophores for each bacterial species. We then coupled ratiometric PCR and automated magnetic beads-based sample preparation within a thermoplastic cartridge and a portable droplet magnetofluidic platform. We named the integrated workflow POC-ratioPCR. We demonstrated that the POC-ratioPCR could detect one out of six bacterial targets related to urinary tract infections (UTIs) in a single reaction using only two-color channels. We further evaluated POC-ratioPCR using mock bacterial urine samples spiked with good agreement. The POC-ratioPCR presents a simple and effective method for enabling broad-based POC PCR identification of pathogens directly from crude biosamples with low optical instrumentation complexity.

Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens.

The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.

A Portable Droplet Magnetofluidic Device for Point-of-Care Detection of Multidrug-Resistant Candida auris.

Candida auris is an emerging multidrug-resistant fungal pathogen that can cause severe and deadly infections. To date, C. auris has spurred outbreaks in healthcare settings in thirty-three countries across five continents. To control and potentially prevent its spread, there is an urgent need for point-of-care (POC) diagnostics that can rapidly screen patients, close patient contacts, and surveil environmental sources. Droplet magnetofluidics (DM), which leverages nucleic acid-binding magnetic beads for realizing POC-amenable nucleic acid detection platforms, offers a promising solution. Herein, we report the first DM device-coined POC.auris-for POC detection of C. auris. As part of POC.auris, we have incorporated a handheld cell lysis module that lyses C. auris cells with 2 min hands-on time. Subsequently, within the palm-sized and automated DM device, C. auris and control DNA are magnetically extracted and purified by a motorized magnetic arm and finally amplified via a duplex real-time quantitative PCR assay by a miniaturized rapid PCR module and a miniaturized fluorescence detector-all in ≤30 min. For demonstration, we use POC.auris to detect C. auris isolates from 3 major clades, with no cross reactivity against other Candida species and a limit of detection of ∼300 colony forming units per mL. Taken together, POC.auris presents a potentially useful tool for combating C. auris.

Filtration-assisted magnetofluidic cartridge platform for HIV RNA detection from blood.

The ability to detect and quantify HIV RNA in blood is essential to sensitive detection of infections and monitoring viremia throughout treatment. Current options for point-of-care HIV diagnosis (i.e. lateral flow rapid tests) lack sensitivity for early detection and are unable to quantify viral load. HIV RNA diagnostics typically require extensive pre-processing of blood to isolate plasma and extract nucleic acids, in addition to expensive equipment for conducting nucleic acid amplification and fluorescence detection. Therefore, molecular HIV diagnostics is still mainly limited to clinical laboratories and there is an unmet need for high sensitivity point-of-care screening and at-home HIV viral load quantification. In this work, we outline a streamlined workflow for extraction of plasma from whole blood coupled with HIV RNA extraction and quantitative polymerase chain reaction (qPCR) in a portable magnetofluidic cartridge platform for use at the point-of-care. Viral particles were isolated from blood using manual filtration through a 3D-printed filter module in seconds followed by automated nucleic acid capture, purification, and transfer to qPCR using magnetic beads. Both nucleic acid extraction and qPCR were integrated within cartridges using compact instrumentation consisting of a motorized magnet arm, miniaturized thermocycler, and image-based fluorescence detection. We demonstrated detection down to 1000 copies of HIV viral particles from whole blood in <30 minutes.

Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing.

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity. Here, we describe the development of a rapid and sensitive magnetofluidic immuno-PCR platform that can address the current gap in point-of-care serological testing for COVID-19. Our magnetofluidic immuno-PCR platform automates a magnetic bead-based, single-binding, and one-wash immuno-PCR assay in a palm-sized magnetofluidic device and delivers results in ∼30 min. In the device, a programmable magnetic arm attracts and transports magnetically-captured antibodies through assay reagents pre-loaded in a companion plastic cartridge, and a miniaturized thermocycler and a fluorescence detector perform immuno-PCR to detect the antibodies. We evaluated our magnetofluidic immuno-PCR with 108 clinical serum/plasma samples and achieved 93.8% (45/48) sensitivity and 98.3% (59/60) specificity, demonstrating its potential as a rapid and sensitive point-of-care serological test for COVID-19.

High resolution estimates of relative gene abundance with quantitative ratiometric regression PCR (qRR-PCR).

Quantification of the relative abundance of genetic traits has broad applications for biomarker discovery, diagnostics, and assessing gene expression in biological research. Relative quantification of genes is traditionally done with the 2-ΔΔCT method using quantitative real-time polymerase chain reaction (qPCR) data, which is often limited in resolution beyond orders of magnitude difference. The latest techniques for quantification of nucleic acids employ digital PCR or microarrays which involve lengthy sample preparation and complex instrumentation. In this work, we describe a quantitative ratiometric regression PCR (qRR-PCR) method for computing relative abundance of genetic traits in a sample with high resolution from a single duplexed real-time quantitative PCR assay. Instead of comparing the individual cycle threshold (Ct) values as is done for the 2-ΔΔCT method, our qRR-PCR algorithm leverages the innate relationship of co-amplified PCR targets to measure their relative quantities using characteristic curves derived from the normalized ratios of qPCR fluorescence curves. We demonstrate the utility of this technique for discriminating the fractional abundance of mixed alleles with resolution below 5%.

Portable Magnetofluidic Device for Point-of-Need Detection of African Swine Fever.

With a nearly 100% mortality rate, African swine fever (ASF) has devastated the pork industry in many countries. Without a vaccine in sight, mitigation rests on rapid diagnosis and immediately depopulating infected or exposed animals. Unfortunately, current tests require centralized laboratories with well-trained personnel, take days to report the results, and thus do not meet the need for such rapid diagnosis. In response, we developed a portable, sample-to-answer device that allows for ASF detection at the point of need in <30 min. The device employs droplet magnetofluidics to automate DNA purification from blood, tissue, or swab samples and utilizes fast thermal cycling to perform real-time quantitative polymerase chain reaction (qPCR), all within an inexpensive disposable cartridge. We evaluated its diagnostic performance at six farms and slaughter facilities. The device exhibits high diagnostic accuracy with a positive percent agreement of 92.2% and a negative percent agreement of 93.6% compared with a lab-based reference qPCR test.

Point-of-care CRISPR-Cas-assisted SARS-CoV-2 detection in an automated and portable droplet magnetofluidic device.

In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but their lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR - a single-step CRISPR-Cas-assisted assay that incoporates sample preparation with minimal manual operation via facile magnetic-based nucleic acid concentration and transport. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/µL SARS-CoV-2 RNA from a sample volume of 100 µL in < 30 min. When evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (Cq values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (Cq < 25) in 20 min.

Magnetofluidic platform for rapid multiplexed screening of SARS-CoV-2 variants and respiratory pathogens.

The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses in the fight to end the pandemic. Widespread detection of variant strains will be critical to inform policy decisions to mitigate further spread, and post-pandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, we have developed a portable, magnetofluidic cartridge platform for automated PCR testing in <30 min. Cartridges were designed for multiplexed detection of SARS-CoV-2 with either distinctive variant mutations or with Influenza A and B. The platform demonstrated a limit of detection down to 2 copies/µL SARS-CoV-2 RNA with successful identification of B.1.1.7 and B.1.351 variants. The multiplexed SARS-CoV-2/Flu assay was validated using archived clinical nasopharyngeal swab eluates ( n = 116) with an overall sensitivity/specificity of 98.1%/95.2%, 85.7%/100%, 100%/98.2%, respectively, for SARS-CoV-2, Influenza A, and Influenza B. Further testing with saliva ( n = 14) demonstrated successful detection of all SARS-CoV-2 positive samples with no false-positives.

A portable magnetofluidic platform for detecting sexually transmitted infections and antimicrobial susceptibility.

Effective treatment of sexually transmitted infections (STIs) is limited by diagnostics that cannot deliver results rapidly while the patient is still in the clinic. The gold standard methods for identification of STIs are nucleic acid amplification tests (NAATs), which are too expensive for widespread use and have lengthy turnaround times. To address the need for fast and affordable diagnostics, we have developed a portable, rapid, on-cartridge magnetofluidic purification and testing (PROMPT) polymerase chain reaction (PCR) test. We show that it can detect Neisseria gonorrhoeae, the pathogen causing gonorrhea, with simultaneous genotyping of the pathogen for resistance to the antimicrobial drug ciprofloxacin in <15 min. The duplex test was integrated into a low-cost thermoplastic cartridge with automated processing of penile swab samples from patients using magnetic beads. A compact instrument conducted DNA extraction, PCR, and analysis of results while relaying data to the user via a smartphone app. This platform was tested on penile swab samples from sexual health clinics in Baltimore, MD, USA (n = 66) and Kampala, Uganda (n = 151) with an overall sensitivity and specificity of 97.7% (95% CI, 94.7 to 100%) and 97.6% (95% CI, 94.1 to 100%), respectively, for N. gonorrhoeae detection and 100% concordance with culture results for ciprofloxacin resistance. This study paves the way for delivering accessible PCR diagnostics for rapidly detecting STIs at the point of care, helping to guide treatment decisions and combat the rise of antimicrobial resistant pathogens.

Digital CRISPR/Cas-Assisted Assay for Rapid and Sensitive Detection of SARS-CoV-2.

The unprecedented demand for rapid diagnostics in response to the COVID-19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)-assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital-based detection. Thus motivated, the first digital CRISPR/Cas-assisted assay-coined digitization-enhanced CRISPR/Cas-assisted one-pot virus detection (deCOViD)-is developed and applied toward SARS-CoV-2 detection. deCOViD is realized through tuning and discretizing a one-step, fluorescence-based, CRISPR/Cas12a-assisted reverse transcription recombinase polymerase amplification assay into sub-nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal-to-background ratio, broad dynamic range, and high sensitivity-down to 1 genome equivalent (GE) µL-1 of SARS-CoV-2 RNA and 20 GE µL-1 of heat-inactivated SARS-CoV-2, which outstrips its benchtop-based counterpart and represents one of the fastest and most sensitive CRISPR/Cas-assisted SARS-CoV-2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas-assisted assays and combating the COVID-19 pandemic and beyond.

Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation for Digital Nucleic Acid Amplification Testing and Analysis.

Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital polymerase chain reaction (PCR), have become useful clinical diagnostic tools. However, nucleic acid (NA) sample preparation preceding dNAAT is generally laborious and performed manually, thus creating the need for a simple sample preparation technique and a facile coupling strategy for dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of NAs with a one-step transfer to dNAAT. Specifically, we leverage droplet magnetofluidics (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for NA extraction and purification. Importantly, the buffer typically used to elute the NAs off the magnetic beads is replaced by a carefully selected PCR solution, enabling direct transfer from sample preparation to dNAAT. Moreover, we demonstrate the potential for multiplexing using a digital high-resolution melt (dHRM) after the digital PCR (dPCR). The utility of this workflow is demonstrated with duplexed detection of bacteria in a sample imitating a coinfection. We first purify the bacterial DNA into a PCR solution using our DM-based sample preparation. We then transfer the purified bacterial DNA to our microfluidic nanoarray to amplify 16S rRNA using dPCR and then perform dHRM to identify the two bacterial species.

Sample-to-Answer Droplet Magnetofluidic Platform for Point-of-Care Hepatitis C Viral Load Quantitation.

Gold standard quantitative nucleic acid tests for diagnosis of viral diseases are currently limited to implementation in laboratories outside of the clinic. An instrument for conducting nucleic acid testing at the point-of-care (POC) that is easily operable by the clinician would reduce the required number of visits to the clinic and improve patient retention for proper treatment. Here we present a droplet magnetofluidic (DM) platform, which leverages functionalized magnetic particles to miniaturize and automate laboratory assays for use in the clinic at the POC. Our novel thermoformed disposable cartridge coupled to a portable multiaxial magnetofluidic instrument enables real-time PCR assays for quantitative and sensitive detection of nucleic acids from crude biosamples. Instead of laborious benchtop sample purification techniques followed by elution and spiking into PCR buffer, the user simply injects the biosample of interest into a cartridge with magnetic particles and loads the cartridge into the instrument. We demonstrate the utility of our platform with hepatitis C virus (HCV) RNA viral load quantitation from blood serum in approximately 1 hour. Clinical serum samples (n = 18) were directly processed on cartridges with no false positives and a limit of detection of 45 IU per 10 µl sample injection.

Thermal Unthreading of the Lasso Peptides Astexin-2 and Astexin-3.

Lasso peptides are a class of knot-like polypeptides in which the C-terminal tail of the peptide threads through a ring formed by an isopeptide bond between the N-terminal amine group and a side chain carboxylic acid. The small size (∼20 amino acids) and simple topology of lasso peptides make them a good model system for studying the unthreading of entangled polypeptides, both with experiments and atomistic simulation. Here, we present an in-depth study of the thermal unthreading behavior of two lasso peptides astexin-2 and astexin-3. Quantitative kinetics and energetics of the unthreading process were determined for variants of these peptides using a series of chromatography and mass spectrometry experiments and biased molecular dynamics (MD) simulations. In addition, we show that the Tyr15Phe variant of astexin-3 unthreads via an unprecedented "tail pulling" mechanism. MD simulations on a model ring-thread system coupled with machine learning approaches also led to the discovery of physicochemical descriptors most important for peptide unthreading.