@article{Tjandra2023,

title = {Rapid Molecular Phenotypic Antimicrobial Susceptibility Test for Neisseria gonorrhoeae Based on Propidium Monoazide Viability PCR},

author = {KC. Tjandra and N. Ram-Mohan and R. Abe and TH. Wang and S. Yang},

url = {https://pubmed.ncbi.nlm.nih.gov/37115656/},

doi = {doi: 10.1021/acsinfecdis.3c00096.},

year  = {2023},

date = {2023-05-12},

journal = {ACS Infectious Diseases},

volume = {9},

issue = {5},

pages = {1160-1167},

abstract = {Neisseria gonorrhoeae (NG) is an urgent threat to antimicrobial resistance (AMR) worldwide. NG has acquired rapid resistance to all previously recommended treatments, leaving ceftriaxone monotherapy as the first and last line of therapy for uncomplicated NG. The ability to rapidly determine susceptibility, which is currently nonexistent for NG, has been proposed as a strategy to preserve ceftriaxone by using alternative treatments. Herein, we used a DNA-intercalating dye in combination with NG-specific primers/probes to generate qPCR cycle threshold (Ct) values at different concentrations of 2 NG-relevant antimicrobials. Our proof-of-concept dual-antimicrobial logistic regression model based on the differential Ct measurements achieved an AUC of 0.93 with a categorical agreement for the susceptibility of 84.6%. When surveying the performance against each antimicrobial separately, the model predicted 90 and 75% susceptible and resistant strains, respectively, to ceftriaxone and 66.7 and 83.3% susceptible and resistant strains, respectively, to ciprofloxacin. We further validated the model against the individual replicates and determined the accuracy of the model in classifying susceptibility agnostic of the inoculum size. We demonstrated a novel PCR-based approach to determine phenotypic ciprofloxacin and ceftriaxone susceptibility information for NG with reasonable accuracy within 30 min, a significant improvement compared to the conventional method which could take multiple days.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2023,

title = {Point-of-Care Amenable Detection of Mycoplasma genitalium and Its Antibiotic Resistance Mutations},

author = {F. Chen and J. Wang and AH. Nambiar and J. Hardick and J. Melendez and AY. Trick and TH. Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/36961769/},

doi = {DOI: 10.1021/acssensors.2c02630},

year  = {2023},

date = {2023-04-28},

journal = {ACS Sensors},

volume = {8},

issue = {4},

pages = {1550-1557},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhao2023,

title = {Multiplex Digital Methylation‐Specific PCR for Noninvasive Screening of Lung Cancer},

author = {Y. Zhao and CM. O'Keefe and K. Hsieh and L. Cope and SC. Joyce and TR. Pisanic and JG. Herman and TH. Wang },

url = {https://pubmed.ncbi.nlm.nih.gov/37039321/},

year  = {2023},

date = {2023-04-11},

journal = {Advanced Science},

volume = {e2206518},

abstract = {There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhu2023,

title = {Dectin-1 signaling on colonic γδ T cells promotes psychosocial stress responses},

author = {X. Zhu and S. Sakamoto and C. Ishii and MD. Smith and K. Ito and M. Obayashi and L. Unger and Y. Hasegawa and S. Kurokawa and T. Kishimoto and H. Li and S. Hatano and TH. Wang and Y. Yoshikai and S. Kano and S. Fukuda and K. Sanada and PA. Calabresi and A. Kamiya

},

url = {https://pubmed.ncbi.nlm.nih.gov/36941398/},

doi = {DOI: 10.1038/s41590-023-01447-8},

year  = {2023},

date = {2023-03-20},

journal = {Nature Immunology},

volume = {24},

pages = {625-636},

abstract = {The intestinal immune system interacts with commensal microbiota to maintain gut homeostasis. Furthermore, stress alters the microbiome composition, leading to impaired brain function; yet how the intestinal immune system mediates these effects remains elusive. Here we report that colonic γδ T cells modulate behavioral vulnerability to chronic social stress via dectin-1 signaling. We show that reduction in specific Lactobacillus species, which are involved in T cell differentiation to protect the host immune system, contributes to stress-induced social-avoidance behavior, consistent with our observations in patients with depression. Stress-susceptible behaviors derive from increased differentiation in colonic interleukin (IL)-17-producing γδ T cells (γδ17 T cells) and their meningeal accumulation. These stress-susceptible cellular and behavioral phenotypes are causally mediated by dectin-1, an innate immune receptor expressed in γδ T cells. Our results highlight the previously unrecognized role of intestinal γδ17 T cells in the modulation of psychological stress responses and the importance of dectin-1 as a potential therapeutic target for the treatment of stress-induced behaviors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Shao2023,

title = {Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucle-ic Acid Amplification Testing Assays},

author = {F. Shao and JS. Park and G. Zhao, and K. Hsieh and TH. Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/36745596/},

doi = {DOI: 10.1021/acs.analchem.2c05632},

year  = {2023},

date = {2023-02-21},

journal = {Analytical Chemistry},

volume = {95},

issue = {7},

pages = {3873-3882},

abstract = {Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Li2023,

title = {Single-cell pathogen diagnostics for combating antibiotic resistance},

author = {H. Li and K. Hsieh and P. Wong and K. Mach and J. Liao and TH. Wang},

url = {https://www.nature.com/articles/s43586-023-00201-6},

doi = {DOI: 10.1021/acs.accounts.1c00462},

year  = {2023},

date = {2023-02-02},

journal = {Nature Reviews Methods Primers},

volume = {3},

number = {6},

issue = {1},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ngo2023b,

title = {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},

author = {HT. Ngo and M. Jin and AY. Trick and FE. Chen and L. Chen and K. Hsieh and TH. Wang

},

url = {https://pubmed.ncbi.nlm.nih.gov/36562405/},

doi = {DOI: 10.1021/acs.analchem.2c03897},

year  = {2023},

date = {2023-01-17},

journal = { Analytical Chemistry},

volume = {95},

issue = {2},

pages = {1159–1168},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Akarapipad2022,

title = {Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19},

author = {P. Akarapipad and E. Bertelson and A. Pessell and TH. Wang and K. Hsieh

},

url = {https://pubmed.ncbi.nlm.nih.gov/36354487/},

doi = {DOI: 10.3390/bios12110978},

year  = {2022},

date = {2022-11-07},

urldate = {2022-11-07},

journal = {Biosensors},

volume = {12},

issue = {11},

pages = {978},

abstract = {The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Li2022,

title = {Payload distribution and capacity of mRNA lipid nanoparticles},

author = {S. Li and Y. Hu and A. Li and J. Lin and K. Hsieh and Z. Schneiderman and P. Zhang and Y. Zhu and C. Qiu and E. Kokkoli and TH. Wang and HQ. Mao},

doi = {https://doi.org/10.1038/s41467-022-33157-4},

year  = {2022},

date = {2022-09-23},

journal = {nature communications},

volume = {13},

number = {5561},

abstract = {Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%–80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2022,

title = {Highly Sensitive Serum Protein Analysis Using Magnetic Bead-Based Proximity Extension Assay},

author = {P. Zhang and J. Hu and JS. Park and K. Hsieh and L. Chen and A. Mao and TH. Wang},

url = {https://pubs.acs.org/doi/full/10.1021/acs.analchem.2c02684},

doi = {https://doi.org/10.1021/acs.analchem.2c02684},

year  = {2022},

date = {2022-08-30},

urldate = {2022-08-30},

journal = {Analytical Chemistry},

volume = {94},

issue = {36},

pages = {1281-12489},

abstract = {Many protein biomarkers are present in biofluids at a very low level but may play critical roles in important biological processes. The fact that these low-abundance proteins remain largely unexplored underscores the importance of developing new tools for highly sensitive protein detection. Although digital enzyme-linked immunosorbent assay (ELISA) has demonstrated ultrahigh sensitivity compared with conventional ELISA, the requirement of specialized instruments limits the accessibility and prevents the widespread implementation. On the other hand, proximity ligation assays (PLA) and proximity extension assays (PEA) show sensitive and specific protein detection using regular laboratory setups, but their sensitivity needs to be further improved to match digital ELISA. To achieve highly sensitive protein detection with minimal accessibility limitation, we develop a magnetic bead-based PEA (magPEA), which posts triple epitope recognition requirement and enables extensive washing for improved sensitivity and enhanced specificity. We demonstrate that the incorporation of magnetic beads into PEA workflow facilitates orders of magnitude sensitivity improvement compared with conventional ELISA, homogeneous PEA, and solid-phase PLA and achieves limits of detection close to that of digital ELISA when using IL-6, IL-8, and GM-CSF as validation. Our magPEA provides a simple approach for highly sensitive protein detection that can be readily implemented to other laboratories and will thus ultimately accelerate the study of the low abundance protein biomarkers in the future.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Facile and scalable tubing-free sample loading for droplet microfluidics},

author = {F. Shao and K. Hsieh and P. Zhang and AM. Kaushik and TH. Wang},

url = {https://www.nature.com/articles/s41598-022-17352-3},

doi = {https://doi.org/10.1038/s41598-022-17352-3},

year  = {2022},

date = {2022-08-03},

journal = {Scientific Reports},

volume = {12},

number = {13340},

issue = {1},

pages = {1-12},

abstract = {Droplet microfluidics has in recent years found a wide range of analytical and bioanalytical applications. In droplet microfluidics, the samples that are discretized into droplets within the devices are predominantly loaded through tubings, but such tubing-based sample loading has drawbacks such as limited scalability for processing many samples, difficulty for automation, and sample wastage. While advances in autosamplers have alleviated some of these drawbacks, sample loading that can instead obviate tubings offers a potentially promising alternative but has been underexplored. To fill the gap, we introduce herein a droplet device that features a new Tubing Eliminated Sample Loading Interface (TESLI). TESLI integrates a network of programmable pneumatic microvalves that regulate vacuum and pressure sources so that successive sub-microliter samples can be directly spotted onto the open-to-atmosphere TESLI inlet, vacuumed into the device, and pressurized into nanoliter droplets within the device with minimal wastage. The same vacuum and pressure regulation also endows TESLI with cleaning and sample switching capabilities, thus enabling scalable processing of many samples in succession. Moreover, we implement a pair of TESLIs in our device to parallelize and alternate their operation as means to minimizing idle time. For demonstration, we use our device to successively process 44 samples into droplets—a number that can further scale. Our results demonstrate the feasibility of tubing-free sample loading and a promising approach for advancing droplet microfluidics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ma2022,

title = {Accurate Nonendoscopic Detection of Esophageal Squamous Cell Carcinoma Using Methylated DNA Biomarkers},

author = {K. Ma and A. Kalra and HL. Tsai and S. Okello and Y. Cheng and SJ. Meltzer and Esophagea Squamous Cell Carcinoma Consortium

},

url = {https://pubmed.ncbi.nlm.nih.gov/35483446/},

doi = {DOI: 10.1053/j.gastro.2022.04.021},

year  = {2022},

date = {2022-08-01},

journal = {Gastroenterology},

volume = { 163},

issue = {2},

pages = {507-509},

abstract = {Esophageal cancer, with 544,076 deaths in 2020, 1 includes esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC). ESCC comprises 90% of esophageal cancers globally. The 5-year survival rate remains poor (15%–25% worldwide) because patients usually present with late disease. 2 Although ESCC can be diagnosed by means of esophagogastroduodenoscopy (EGD) with biopsy, EGD is not widely available in low-income countries. 2 Inexpensive, safe, accessible diagnostic alternatives will likely improve diagnosis and outcome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Shao2022b,

title = {Emerging platforms for high-throughput enzymatic bioassays},

author = {F. Shao and P. Lee and H. Li and K. Hsieh and TH. Wang

},

url = {https://pubmed.ncbi.nlm.nih.gov/35863950/},

doi = {DOI: 10.1016/j.tibtech.2022.06.006},

year  = {2022},

date = {2022-07-18},

journal = {Trends in Biotechnology},

volume = { 41},

issue = {1},

pages = {120-133},

abstract = {Enzymes have essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in vitro enzymatic bioassays have been developed for use in industrial and research fields, such as cell biology, enzyme engineering, drug screening, and biofuel production. Of note, many of these require the use of high-throughput platforms. Although the microtiter plate remains the standard for high-throughput enzymatic bioassays, microfluidic arrays and droplet microfluidics represent emerging methods. Each has seen significant advances and offers distinct advantages; however, drawbacks in key performance metrics, including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity, remain. Herein, we compare recent high-throughput platforms using the aforementioned metrics as criteria and provide insights into remaining challenges and future research trends.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2022,

title = {Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification},

author = {FE. Chen and AY. Trick and AC. Hasnain and K. Hsieh and L. Chen and DJ. Shin and TH. Wang},

url = {https://pubs.acs.org/doi/full/10.1021/acs.analchem.2c01357},

doi = {https://doi.org/10.1021/acs.analchem.2c01357},

year  = {2022},

date = {2022-06-21},

urldate = {2022-06-21},

journal = {Analytical Chemistry},

volume = {94},

issue = {26},

pages = {9372-9379},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Liberto2022,

title = {Current and Emerging Methods for Ovarian Cancer Screening and Diagnostics: A Comprehensive Review},

author = {JM. Liberto and SY. Chen and IM. Shih and TH. Wang and TL. Wang and TR. Pisanic},

url = {https://pubmed.ncbi.nlm.nih.gov/35740550/},

doi = {DOI: 10.3390/cancers14122885},

year  = {2022},

date = {2022-06-11},

journal = {Cancers},

volume = {14},

number = {2885},

issue = {12},

abstract = {With a 5-year survival rate of less than 50%, ovarian high-grade serous carcinoma (HGSC) is one of the most highly aggressive gynecological malignancies affecting women today. The high mortality rate of HGSC is largely attributable to delays in diagnosis, as most patients remain undiagnosed until the late stages of -disease. There are currently no recommended screening tests for ovarian cancer and there thus remains an urgent need for new diagnostic methods, particularly those that can detect the disease at early stages when clinical intervention remains effective. While diagnostics for ovarian cancer share many of the same technical hurdles as for other cancer types, the low prevalence of the disease in the general population, coupled with a notable lack of sensitive and specific biomarkers, have made the development of a clinically useful screening strategy particularly challenging. Here, we present a detailed review of the overall landscape of ovarian cancer diagnostics, with emphasis on emerging methods that employ novel protein, genetic, epigenetic and imaging-based biomarkers and/or advanced diagnostic technologies for the noninvasive detection of HGSC, particularly in women at high risk due to germline mutations such as BRCA1/2. Lastly, we discuss the translational potential of these approaches for achieving a clinically implementable solution for screening and diagnostics of early-stage ovarian cancer as a means of ultimately improving patient outcomes in both the general and high-risk populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Stark2022,

title = {High-throughput sample processing for methylation analysis in an automated, enclosed environment},

author = {A. Stark and TR. Pisanic II and JG. Herman and TH. Wang },

url = {https://www.sciencedirect.com/science/article/pii/S2472630321000248},

doi = {https://doi.org/10.1016/j.slast.2021.12.002},

year  = {2022},

date = {2022-06-01},

journal = {SLAS Technology},

volume = {27},

issue = {3},

pages = {172-179},

abstract = {Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated. Thus, sample processing methods still require numerous manual steps that may reduce sample throughput and precision, while increasing the risk of contamination and human error. In this work, we present an integrated, automated solution for performing the entire sample preparation process, including DNA extraction, purification, bisulfite conversion and PCR plate preparation within in an enclosed environment. The method employs silica-coated magnetic particles that eliminate the need for a centrifuge or vacuum manifold, thereby reducing the complexity and cost of the required automation platform. Toward this end, we also compare commercial DNA extraction and bisulfite conversion kits to identify a protocol suitable for automation to significantly improve genomic and bisulfite-treated DNA yields over manufacturer protocols. Overall, this research demonstrated development of an automated protocol that offers the ability to generate high-quality, bisulfite-treated DNA samples in a high-throughput and clean environment with minimal user intervention and comparable yields to manual processing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hamill2022b,

title = {High burden of untreated syphilis, drug resistant Neisseria gonorrhoeae, and other sexually transmitted infections in men with urethral discharge syndrome in Kampala, Uganda},

author = {MM. Hamill and A. Onzia and TH. Wang and AN. Kiragga and YH. Hsieh and R. Parkes-Ratanshi and E. Gough and P. Kyambadde and JH. Melendez and YC. Manabe

},

url = {https://pubmed.ncbi.nlm.nih.gov/35525934/},

doi = {DOI: 10.1186/s12879-022-07431-1},

year  = {2022},

date = {2022-05-07},

journal = {BMC Infectious Diseases},

volume = { 22},

issue = {1},

pages = {1-9},

abstract = {Objectives: Prompt diagnosis and treatment of sexually transmitted infections (STIs) are essential to combat the STI epidemic in resource-limited settings. We characterized the burden of 5 curable STIs chlamydia, gonorrhea, trichomoniasis, Mycoplasma genitalium, syphilis, and HIV infection in Ugandan men with urethritis.



Methods: Participants were recruited from a gonococcal surveillance program in Kampala, Uganda. Questionnaires, penile swabs were collected and tested by nucleic acid amplification. Gonococcal isolates were tested for antimicrobial sensitivity. Sequential point-of-care tests on blood samples were used to screen for syphilis and HIV. Bivariable and multivariable multinomial logistic regression models were used to estimate odds ratios for preselected factors likely to be associated with STIs. Adherence to STI treatment guidelines were analyzed.



Results: From October 2019 to November 2020, positivity (95% CI) for gonorrhea, chlamydia, trichomoniasis, and Mycoplasma genitalium, were 66.4% (60.1%, 72.2%), 21.7% (16.8%, 27.4%), 2.0% (0.7%, 4.9%), and 12.4% (8.7%, 17.3%) respectively. All Neisseria gonorrhoeae isolates were resistant to ciprofloxacin, penicillin, and tetracycline, but susceptible to extended spectrum cephalosporins and azithromycin. HIV and syphilis prevalence was 20.0% (50/250) and 10.0% (25/250), and the proportion unaware of their infection was 4.0% and 80.0% respectively. Most participants were treated per national guidelines. Multivariable analysis demonstrated significant associations between curable STI coinfections and younger age, transactional sex, but not HIV status, nor condom or alcohol use.



Conclusions: STI coinfections including HIV their associated risk factors, and gonococcal AMR were common in this population. The majority with syphilis were unaware of their infection and were untreated. Transactional sex was associated with STI coinfections, and > 80% of participants received appropriate treatment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lee2022,

title = {A portable droplet magnetofluidic device for point-of-care detection of multidrug-resistant Candida auris},

author = {PW. Lee and M. Totten and L. Chen and FE. Chen and AY. Trick and K. Shah and HT. Ngo and M. Jin and K. Hsieh and SX. Zhang and TH. Wang},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9003015/},

doi = {DOI: 10.3389/fbioe.2022.826694},

year  = {2022},

date = {2022-03-24},

journal = {Frontiers in Bioengineering and Biotechnology},

volume = {10},

number = {926694},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Trick2022,

title = {Filtration-assisted magnetofluidic cartridge platform for HIV RNA detection from blood},

author = {A.Y. Trick and H.T. Ngo and AH. Nambiar and MM. Morakis and FE. Chen and L. Chen and K. Hsieh and T.H. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2022/lc/d1lc00820j},

doi = {https://doi.org/10.1039/D1LC00820J},

year  = {2022},

date = {2022-01-24},

urldate = {2022-01-24},

journal = {Lab on a Chip},

volume = {22},

pages = {945-953},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Tjandra2023,

title = {Rapid Molecular Phenotypic Antimicrobial Susceptibility Test for Neisseria gonorrhoeae Based on Propidium Monoazide Viability PCR},

author = {KC. Tjandra and N. Ram-Mohan and R. Abe and TH. Wang and S. Yang},

url = {https://pubmed.ncbi.nlm.nih.gov/37115656/},

doi = {doi: 10.1021/acsinfecdis.3c00096.},

year  = {2023},

date = {2023-05-12},

journal = {ACS Infectious Diseases},

volume = {9},

issue = {5},

pages = {1160-1167},

abstract = {Neisseria gonorrhoeae (NG) is an urgent threat to antimicrobial resistance (AMR) worldwide. NG has acquired rapid resistance to all previously recommended treatments, leaving ceftriaxone monotherapy as the first and last line of therapy for uncomplicated NG. The ability to rapidly determine susceptibility, which is currently nonexistent for NG, has been proposed as a strategy to preserve ceftriaxone by using alternative treatments. Herein, we used a DNA-intercalating dye in combination with NG-specific primers/probes to generate qPCR cycle threshold (Ct) values at different concentrations of 2 NG-relevant antimicrobials. Our proof-of-concept dual-antimicrobial logistic regression model based on the differential Ct measurements achieved an AUC of 0.93 with a categorical agreement for the susceptibility of 84.6%. When surveying the performance against each antimicrobial separately, the model predicted 90 and 75% susceptible and resistant strains, respectively, to ceftriaxone and 66.7 and 83.3% susceptible and resistant strains, respectively, to ciprofloxacin. We further validated the model against the individual replicates and determined the accuracy of the model in classifying susceptibility agnostic of the inoculum size. We demonstrated a novel PCR-based approach to determine phenotypic ciprofloxacin and ceftriaxone susceptibility information for NG with reasonable accuracy within 30 min, a significant improvement compared to the conventional method which could take multiple days.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2023,

title = {Point-of-Care Amenable Detection of Mycoplasma genitalium and Its Antibiotic Resistance Mutations},

author = {F. Chen and J. Wang and AH. Nambiar and J. Hardick and J. Melendez and AY. Trick and TH. Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/36961769/},

doi = {DOI: 10.1021/acssensors.2c02630},

year  = {2023},

date = {2023-04-28},

journal = {ACS Sensors},

volume = {8},

issue = {4},

pages = {1550-1557},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhao2023,

title = {Multiplex Digital Methylation‐Specific PCR for Noninvasive Screening of Lung Cancer},

author = {Y. Zhao and CM. O'Keefe and K. Hsieh and L. Cope and SC. Joyce and TR. Pisanic and JG. Herman and TH. Wang },

url = {https://pubmed.ncbi.nlm.nih.gov/37039321/},

year  = {2023},

date = {2023-04-11},

journal = {Advanced Science},

volume = {e2206518},

abstract = {There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhu2023,

title = {Dectin-1 signaling on colonic γδ T cells promotes psychosocial stress responses},

author = {X. Zhu and S. Sakamoto and C. Ishii and MD. Smith and K. Ito and M. Obayashi and L. Unger and Y. Hasegawa and S. Kurokawa and T. Kishimoto and H. Li and S. Hatano and TH. Wang and Y. Yoshikai and S. Kano and S. Fukuda and K. Sanada and PA. Calabresi and A. Kamiya

},

url = {https://pubmed.ncbi.nlm.nih.gov/36941398/},

doi = {DOI: 10.1038/s41590-023-01447-8},

year  = {2023},

date = {2023-03-20},

journal = {Nature Immunology},

volume = {24},

pages = {625-636},

abstract = {The intestinal immune system interacts with commensal microbiota to maintain gut homeostasis. Furthermore, stress alters the microbiome composition, leading to impaired brain function; yet how the intestinal immune system mediates these effects remains elusive. Here we report that colonic γδ T cells modulate behavioral vulnerability to chronic social stress via dectin-1 signaling. We show that reduction in specific Lactobacillus species, which are involved in T cell differentiation to protect the host immune system, contributes to stress-induced social-avoidance behavior, consistent with our observations in patients with depression. Stress-susceptible behaviors derive from increased differentiation in colonic interleukin (IL)-17-producing γδ T cells (γδ17 T cells) and their meningeal accumulation. These stress-susceptible cellular and behavioral phenotypes are causally mediated by dectin-1, an innate immune receptor expressed in γδ T cells. Our results highlight the previously unrecognized role of intestinal γδ17 T cells in the modulation of psychological stress responses and the importance of dectin-1 as a potential therapeutic target for the treatment of stress-induced behaviors.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Shao2023,

title = {Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucle-ic Acid Amplification Testing Assays},

author = {F. Shao and JS. Park and G. Zhao, and K. Hsieh and TH. Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/36745596/},

doi = {DOI: 10.1021/acs.analchem.2c05632},

year  = {2023},

date = {2023-02-21},

journal = {Analytical Chemistry},

volume = {95},

issue = {7},

pages = {3873-3882},

abstract = {Developing assays that combine CRISPR/Cas and isothermal nucleic acid amplification has become a burgeoning research area due to the novelty and simplicity of CRISPR/Cas and the potential for point-of-care uses. Most current research explores various two-step assays by appending different CRISPR/Cas effectors to the end of different isothermal nucleic acid amplification methods. However, efforts in integrating both components into more ideal single-step assays are scarce, and poor-performing single-step assays have been reported. Moreover, lack of investigations into CRISPR/Cas in single-step assays results in incomplete understanding. To fill this knowledge gap, we conducted a systematic investigation by developing and comparing assays that share the identical recombinase polymerase amplification (RPA) but differ in CRISPR/Cas12a. We found that the addition of CRISPR/Cas12a indeed unlocks signal amplification but, at the same time, impedes RPA and that CRISPR/Cas12a concentration is a key parameter for attenuating RPA impediment and ensuring assay performance. Accordingly, we found that our protospacer adjacent motif (PAM)-free CRISPR/Cas12a-assisted RPA assay, which only moderately impeded RPA at its optimal CRISPR/Cas12a concentration, outperformed its counterparts in assay design, signal, sensitivity, and speed. We also discovered that a new commercial Cas12a effector could also drive our PAM-free CRISPR/Cas12a-assisted RPA assay and reduce its cost, though simultaneously lowering its signal. Our study and the new insights can be broadly applied to steer and facilitate further advances in CRISPR/Cas-based assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Li2023,

title = {Single-cell pathogen diagnostics for combating antibiotic resistance},

author = {H. Li and K. Hsieh and P. Wong and K. Mach and J. Liao and TH. Wang},

url = {https://www.nature.com/articles/s43586-023-00201-6},

doi = {DOI: 10.1021/acs.accounts.1c00462},

year  = {2023},

date = {2023-02-02},

journal = {Nature Reviews Methods Primers},

volume = {3},

number = {6},

issue = {1},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ngo2023b,

title = {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},

author = {HT. Ngo and M. Jin and AY. Trick and FE. Chen and L. Chen and K. Hsieh and TH. Wang

},

url = {https://pubmed.ncbi.nlm.nih.gov/36562405/},

doi = {DOI: 10.1021/acs.analchem.2c03897},

year  = {2023},

date = {2023-01-17},

journal = { Analytical Chemistry},

volume = {95},

issue = {2},

pages = {1159–1168},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Akarapipad2022,

title = {Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19},

author = {P. Akarapipad and E. Bertelson and A. Pessell and TH. Wang and K. Hsieh

},

url = {https://pubmed.ncbi.nlm.nih.gov/36354487/},

doi = {DOI: 10.3390/bios12110978},

year  = {2022},

date = {2022-11-07},

urldate = {2022-11-07},

journal = {Biosensors},

volume = {12},

issue = {11},

pages = {978},

abstract = {The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Li2022,

title = {Payload distribution and capacity of mRNA lipid nanoparticles},

author = {S. Li and Y. Hu and A. Li and J. Lin and K. Hsieh and Z. Schneiderman and P. Zhang and Y. Zhu and C. Qiu and E. Kokkoli and TH. Wang and HQ. Mao},

doi = {https://doi.org/10.1038/s41467-022-33157-4},

year  = {2022},

date = {2022-09-23},

journal = {nature communications},

volume = {13},

number = {5561},

abstract = {Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%–80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2022,

title = {Highly Sensitive Serum Protein Analysis Using Magnetic Bead-Based Proximity Extension Assay},

author = {P. Zhang and J. Hu and JS. Park and K. Hsieh and L. Chen and A. Mao and TH. Wang},

url = {https://pubs.acs.org/doi/full/10.1021/acs.analchem.2c02684},

doi = {https://doi.org/10.1021/acs.analchem.2c02684},

year  = {2022},

date = {2022-08-30},

urldate = {2022-08-30},

journal = {Analytical Chemistry},

volume = {94},

issue = {36},

pages = {1281-12489},

abstract = {Many protein biomarkers are present in biofluids at a very low level but may play critical roles in important biological processes. The fact that these low-abundance proteins remain largely unexplored underscores the importance of developing new tools for highly sensitive protein detection. Although digital enzyme-linked immunosorbent assay (ELISA) has demonstrated ultrahigh sensitivity compared with conventional ELISA, the requirement of specialized instruments limits the accessibility and prevents the widespread implementation. On the other hand, proximity ligation assays (PLA) and proximity extension assays (PEA) show sensitive and specific protein detection using regular laboratory setups, but their sensitivity needs to be further improved to match digital ELISA. To achieve highly sensitive protein detection with minimal accessibility limitation, we develop a magnetic bead-based PEA (magPEA), which posts triple epitope recognition requirement and enables extensive washing for improved sensitivity and enhanced specificity. We demonstrate that the incorporation of magnetic beads into PEA workflow facilitates orders of magnitude sensitivity improvement compared with conventional ELISA, homogeneous PEA, and solid-phase PLA and achieves limits of detection close to that of digital ELISA when using IL-6, IL-8, and GM-CSF as validation. Our magPEA provides a simple approach for highly sensitive protein detection that can be readily implemented to other laboratories and will thus ultimately accelerate the study of the low abundance protein biomarkers in the future.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Facile and scalable tubing-free sample loading for droplet microfluidics},

author = {F. Shao and K. Hsieh and P. Zhang and AM. Kaushik and TH. Wang},

url = {https://www.nature.com/articles/s41598-022-17352-3},

doi = {https://doi.org/10.1038/s41598-022-17352-3},

year  = {2022},

date = {2022-08-03},

journal = {Scientific Reports},

volume = {12},

number = {13340},

issue = {1},

pages = {1-12},

abstract = {Droplet microfluidics has in recent years found a wide range of analytical and bioanalytical applications. In droplet microfluidics, the samples that are discretized into droplets within the devices are predominantly loaded through tubings, but such tubing-based sample loading has drawbacks such as limited scalability for processing many samples, difficulty for automation, and sample wastage. While advances in autosamplers have alleviated some of these drawbacks, sample loading that can instead obviate tubings offers a potentially promising alternative but has been underexplored. To fill the gap, we introduce herein a droplet device that features a new Tubing Eliminated Sample Loading Interface (TESLI). TESLI integrates a network of programmable pneumatic microvalves that regulate vacuum and pressure sources so that successive sub-microliter samples can be directly spotted onto the open-to-atmosphere TESLI inlet, vacuumed into the device, and pressurized into nanoliter droplets within the device with minimal wastage. The same vacuum and pressure regulation also endows TESLI with cleaning and sample switching capabilities, thus enabling scalable processing of many samples in succession. Moreover, we implement a pair of TESLIs in our device to parallelize and alternate their operation as means to minimizing idle time. For demonstration, we use our device to successively process 44 samples into droplets—a number that can further scale. Our results demonstrate the feasibility of tubing-free sample loading and a promising approach for advancing droplet microfluidics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ma2022,

title = {Accurate Nonendoscopic Detection of Esophageal Squamous Cell Carcinoma Using Methylated DNA Biomarkers},

author = {K. Ma and A. Kalra and HL. Tsai and S. Okello and Y. Cheng and SJ. Meltzer and Esophagea Squamous Cell Carcinoma Consortium

},

url = {https://pubmed.ncbi.nlm.nih.gov/35483446/},

doi = {DOI: 10.1053/j.gastro.2022.04.021},

year  = {2022},

date = {2022-08-01},

journal = {Gastroenterology},

volume = { 163},

issue = {2},

pages = {507-509},

abstract = {Esophageal cancer, with 544,076 deaths in 2020, 1 includes esophageal adenocarcinoma and esophageal squamous cell carcinoma (ESCC). ESCC comprises 90% of esophageal cancers globally. The 5-year survival rate remains poor (15%–25% worldwide) because patients usually present with late disease. 2 Although ESCC can be diagnosed by means of esophagogastroduodenoscopy (EGD) with biopsy, EGD is not widely available in low-income countries. 2 Inexpensive, safe, accessible diagnostic alternatives will likely improve diagnosis and outcome.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Shao2022b,

title = {Emerging platforms for high-throughput enzymatic bioassays},

author = {F. Shao and P. Lee and H. Li and K. Hsieh and TH. Wang

},

url = {https://pubmed.ncbi.nlm.nih.gov/35863950/},

doi = {DOI: 10.1016/j.tibtech.2022.06.006},

year  = {2022},

date = {2022-07-18},

journal = {Trends in Biotechnology},

volume = { 41},

issue = {1},

pages = {120-133},

abstract = {Enzymes have essential roles in catalyzing biological reactions and maintaining metabolic systems. Many in vitro enzymatic bioassays have been developed for use in industrial and research fields, such as cell biology, enzyme engineering, drug screening, and biofuel production. Of note, many of these require the use of high-throughput platforms. Although the microtiter plate remains the standard for high-throughput enzymatic bioassays, microfluidic arrays and droplet microfluidics represent emerging methods. Each has seen significant advances and offers distinct advantages; however, drawbacks in key performance metrics, including reagent consumption, reaction manipulation, reaction recovery, real-time measurement, concentration gradient range, and multiplexity, remain. Herein, we compare recent high-throughput platforms using the aforementioned metrics as criteria and provide insights into remaining challenges and future research trends.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2022,

title = {Ratiometric PCR in a Portable Sample-to-Result Device for Broad-Based Pathogen Identification},

author = {FE. Chen and AY. Trick and AC. Hasnain and K. Hsieh and L. Chen and DJ. Shin and TH. Wang},

url = {https://pubs.acs.org/doi/full/10.1021/acs.analchem.2c01357},

doi = {https://doi.org/10.1021/acs.analchem.2c01357},

year  = {2022},

date = {2022-06-21},

urldate = {2022-06-21},

journal = {Analytical Chemistry},

volume = {94},

issue = {26},

pages = {9372-9379},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Liberto2022,

title = {Current and Emerging Methods for Ovarian Cancer Screening and Diagnostics: A Comprehensive Review},

author = {JM. Liberto and SY. Chen and IM. Shih and TH. Wang and TL. Wang and TR. Pisanic},

url = {https://pubmed.ncbi.nlm.nih.gov/35740550/},

doi = {DOI: 10.3390/cancers14122885},

year  = {2022},

date = {2022-06-11},

journal = {Cancers},

volume = {14},

number = {2885},

issue = {12},

abstract = {With a 5-year survival rate of less than 50%, ovarian high-grade serous carcinoma (HGSC) is one of the most highly aggressive gynecological malignancies affecting women today. The high mortality rate of HGSC is largely attributable to delays in diagnosis, as most patients remain undiagnosed until the late stages of -disease. There are currently no recommended screening tests for ovarian cancer and there thus remains an urgent need for new diagnostic methods, particularly those that can detect the disease at early stages when clinical intervention remains effective. While diagnostics for ovarian cancer share many of the same technical hurdles as for other cancer types, the low prevalence of the disease in the general population, coupled with a notable lack of sensitive and specific biomarkers, have made the development of a clinically useful screening strategy particularly challenging. Here, we present a detailed review of the overall landscape of ovarian cancer diagnostics, with emphasis on emerging methods that employ novel protein, genetic, epigenetic and imaging-based biomarkers and/or advanced diagnostic technologies for the noninvasive detection of HGSC, particularly in women at high risk due to germline mutations such as BRCA1/2. Lastly, we discuss the translational potential of these approaches for achieving a clinically implementable solution for screening and diagnostics of early-stage ovarian cancer as a means of ultimately improving patient outcomes in both the general and high-risk populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Stark2022,

title = {High-throughput sample processing for methylation analysis in an automated, enclosed environment},

author = {A. Stark and TR. Pisanic II and JG. Herman and TH. Wang },

url = {https://www.sciencedirect.com/science/article/pii/S2472630321000248},

doi = {https://doi.org/10.1016/j.slast.2021.12.002},

year  = {2022},

date = {2022-06-01},

journal = {SLAS Technology},

volume = {27},

issue = {3},

pages = {172-179},

abstract = {Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated. Thus, sample processing methods still require numerous manual steps that may reduce sample throughput and precision, while increasing the risk of contamination and human error. In this work, we present an integrated, automated solution for performing the entire sample preparation process, including DNA extraction, purification, bisulfite conversion and PCR plate preparation within in an enclosed environment. The method employs silica-coated magnetic particles that eliminate the need for a centrifuge or vacuum manifold, thereby reducing the complexity and cost of the required automation platform. Toward this end, we also compare commercial DNA extraction and bisulfite conversion kits to identify a protocol suitable for automation to significantly improve genomic and bisulfite-treated DNA yields over manufacturer protocols. Overall, this research demonstrated development of an automated protocol that offers the ability to generate high-quality, bisulfite-treated DNA samples in a high-throughput and clean environment with minimal user intervention and comparable yields to manual processing.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Hamill2022b,

title = {High burden of untreated syphilis, drug resistant Neisseria gonorrhoeae, and other sexually transmitted infections in men with urethral discharge syndrome in Kampala, Uganda},

author = {MM. Hamill and A. Onzia and TH. Wang and AN. Kiragga and YH. Hsieh and R. Parkes-Ratanshi and E. Gough and P. Kyambadde and JH. Melendez and YC. Manabe

},

url = {https://pubmed.ncbi.nlm.nih.gov/35525934/},

doi = {DOI: 10.1186/s12879-022-07431-1},

year  = {2022},

date = {2022-05-07},

journal = {BMC Infectious Diseases},

volume = { 22},

issue = {1},

pages = {1-9},

abstract = {Objectives: Prompt diagnosis and treatment of sexually transmitted infections (STIs) are essential to combat the STI epidemic in resource-limited settings. We characterized the burden of 5 curable STIs chlamydia, gonorrhea, trichomoniasis, Mycoplasma genitalium, syphilis, and HIV infection in Ugandan men with urethritis.



Methods: Participants were recruited from a gonococcal surveillance program in Kampala, Uganda. Questionnaires, penile swabs were collected and tested by nucleic acid amplification. Gonococcal isolates were tested for antimicrobial sensitivity. Sequential point-of-care tests on blood samples were used to screen for syphilis and HIV. Bivariable and multivariable multinomial logistic regression models were used to estimate odds ratios for preselected factors likely to be associated with STIs. Adherence to STI treatment guidelines were analyzed.



Results: From October 2019 to November 2020, positivity (95% CI) for gonorrhea, chlamydia, trichomoniasis, and Mycoplasma genitalium, were 66.4% (60.1%, 72.2%), 21.7% (16.8%, 27.4%), 2.0% (0.7%, 4.9%), and 12.4% (8.7%, 17.3%) respectively. All Neisseria gonorrhoeae isolates were resistant to ciprofloxacin, penicillin, and tetracycline, but susceptible to extended spectrum cephalosporins and azithromycin. HIV and syphilis prevalence was 20.0% (50/250) and 10.0% (25/250), and the proportion unaware of their infection was 4.0% and 80.0% respectively. Most participants were treated per national guidelines. Multivariable analysis demonstrated significant associations between curable STI coinfections and younger age, transactional sex, but not HIV status, nor condom or alcohol use.



Conclusions: STI coinfections including HIV their associated risk factors, and gonococcal AMR were common in this population. The majority with syphilis were unaware of their infection and were untreated. Transactional sex was associated with STI coinfections, and > 80% of participants received appropriate treatment.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Lee2022,

title = {A portable droplet magnetofluidic device for point-of-care detection of multidrug-resistant Candida auris},

author = {PW. Lee and M. Totten and L. Chen and FE. Chen and AY. Trick and K. Shah and HT. Ngo and M. Jin and K. Hsieh and SX. Zhang and TH. Wang},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9003015/},

doi = {DOI: 10.3389/fbioe.2022.826694},

year  = {2022},

date = {2022-03-24},

journal = {Frontiers in Bioengineering and Biotechnology},

volume = {10},

number = {926694},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Trick2022,

title = {Filtration-assisted magnetofluidic cartridge platform for HIV RNA detection from blood},

author = {A.Y. Trick and H.T. Ngo and AH. Nambiar and MM. Morakis and FE. Chen and L. Chen and K. Hsieh and T.H. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2022/lc/d1lc00820j},

doi = {https://doi.org/10.1039/D1LC00820J},

year  = {2022},

date = {2022-01-24},

urldate = {2022-01-24},

journal = {Lab on a Chip},

volume = {22},

pages = {945-953},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Combating Antimicrobial Resistance via Single-Cell Diagnostic Technologies Powered by Droplet Microfluidics},

author = {K. Hsieh and KE. Mach and P. Zhang and JC. Liao and TH. Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/34898173/},

doi = {DOI: 10.1021/acs.accounts.1c00462},

year  = {2022},

date = {2022-01-18},

journal = {Accounts of Chemical Research},

volume = {55},

pages = {123–133},

abstract = {Antimicrobial resistance is a global threat that if left unchecked could lead to 10 million annual mortalities by 2050. One factor contributing to the rise of multi-drug-resistant (MDR) pathogens is the reliance on traditional culture-based pathogen identification (ID) and antimicrobial susceptibility testing (AST) that typically takes several days. This delay of objective pathogen ID and AST information to inform clinical decision making results in clinicians treating patients empirically often using first-line, broad-spectrum antibiotics, contributing to the misuse/overuse of antibiotics. To combat the rise in MDR pathogens, there is a critical demand for rapid ID and AST technologies. Among the advances in ID and AST technologies in the past decade, single-cell diagnostic technologies powered by droplet microfluidics offer great promise due to their potential for high-sensitivity detection and rapid turnaround time. Our laboratory has been at the forefront of developing such technologies and applying them to diagnosing urinary tract infections (UTIs), one of the most common infections and a frequent reason for the prescription of antimicrobials. For pathogen ID, we first demonstrated the highly sensitive, amplification-free detection of single bacterial cells by confining them in picoliter-scale droplets and detection with fluorogenic peptide nucleic acid (PNA) probes that target their 16S rRNA (rRNA), a well-characterized marker for phylogenic classification. We subsequently improved the PNA probe design and enhanced detection sensitivity. For single-cell AST, we first employed a growth indicator dye and engineered an integrated device that allows us to detect growth from single bacterial cells under antibiotic exposure within 1 h, equivalent to two to three bacterial replications. To expand beyond testing a single antibiotic condition per device, a common limitation for droplet microfluidics, we developed an integrated programmable droplet microfluidic device for scalable single-cell AST. Using the scalable single-cell AST platform, we demonstrated the generation of up to 32 droplet groups in a single device with custom antibiotic titers and the capacity to scale up single-cell AST, and providing reliable pathogen categories beyond a binary call embodies a critical advance. Finally, we developed an integrated ID and AST platform. To this end, we developed a PNA probe panel that can identify nearly 90% of uropathogens and showed the quantitative detection of 16S rRNA from single bacterial cells in droplet-enabled AST after as little as 10 min of antibiotic exposure. This platform achieved both ID and AST from minimally processed urine samples in 30 min, representing one of the fastest turnaround times to date. In addition to tracing the development of our technologies, we compare them with contemporary research advances and offer our perspectives for future development, with the vision that single-cell ID and AST technologies powered by droplet microfluidics can indeed become a useful diagnostic tool for combating antimicrobial resistance},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Li2022b,

title = {Combinatorial nanodroplet platform for screening antibiotic combinations},

author = {H. Li and P. Zhang and K. Hsieh and T.H. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2022/lc/d1lc00865j},

doi = {DOI	https://doi.org/10.1039/D1LC00865J},

year  = {2022},

date = {2022-01-11},

journal = {Lab on a Chip},

volume = {22},

pages = {621-631},

abstract = {The emergence and spread of multidrug resistant bacterial strains and concomitant dwindling of effective antibiotics pose worldwide healthcare challenges. To address these challenges, advanced engineering tools are developed to personalize antibiotic treatments by speeding up the diagnostics that is critical to prevent antibiotic misuse and overuse and make full use of existing antibiotics. Meanwhile, it is necessary to investigate novel antibiotic strategies. Recently, repurposing mono antibiotics into combinatorial antibiotic therapies has shown great potential for treatment of bacterial infections. However, widespread adoption of drug combinations has been hindered by the complexity of screening techniques and the cost of reagent consumptions in practice. In this study, we developed a combinatorial nanodroplet platform for automated and high-throughput screening of antibiotic combinations while consuming orders of magnitude lower reagents than the standard microtiter-based screening method. In particular, the proposed platform is capable of creating nanoliter droplets with multiple reagents in an automatic manner, tuning concentrations of each component, performing biochemical assays with high flexibility (e.g., temperature and duration), and achieving detection with high sensitivity. A biochemical assay, based on the reduction of resazurin by the metabolism of bacteria, has been characterized and employed to evaluate the combinatorial effects of the antibiotics of interest. In a pilot study, we successfully screened pairwise combinations between 4 antibiotics for a model Escherichia coli strain.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory Pathogens},

author = {AY. Trick and FE. Chen and L. Chen and PW. Lee and AC. Hasnain and HH. Mostafa and KC. Carroll and TH. Wang},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/admt.202101013},

doi = { https://doi.org/10.1002/admt.202101013},

year  = {2022},

date = {2022-01-07},

journal = {Advanced Materials Technologies},

volume = {7},

number = {2101013},

issue = {6},

abstract = {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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kranenbarg2021,

title = {High performance methylated DNA markers for detection of colon adenocarcinoma},

author = {RAM. Klein Kranenbarg and AH. Vali and JNM. IJzermans and TR. Pisanic and TH. Wang and N. Azad and S. Sukumar and M. Fackler},

url = {https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-021-01206-2},

doi = {https://doi.org/10.1186/s13148-021-01206-2},

year  = {2021},

date = {2021-12-13},

journal = {Clinical Epigenetics},

volume = {13},

issue = {1},

pages = {1-15},

abstract = {Background

Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA.



Methods

Hierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann–Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy.



Results

In the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue.



Conclusions

We identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2021,

title = {A Cascaded Droplet Microfluidic Platform Enables High-throughput Single Cell Antibiotic Susceptibility Testing at Scale},

author = {P. Zhang and A. Kaushik and K. Hsieh and S. Li and S. Lewis and K.E. Mach and .C. Liao and K.C. Carroll},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/smtd.202101254},

doi = {https://doi.org/10.1101/2021.06.25.21259551},

year  = {2021},

date = {2021-12-08},

urldate = {2021-07-02},

journal = {Small Methods},

volume = {6},

issue = {1},

abstract = {The global threat of antibiotic resistance underscores critical but unmet needs for rapid antibiotic susceptibility testing (AST) technologies. To this end, droplet microfluidic-based single-cell AST offers promise by achieving unprecedented rapidity, but its potential for clinical use is marred by the capacity of testing one to few antibiotic conditions per device, which falls short from the required scale in clinically relevant scenarios. To lift the scalability constraint in rapid single-cell AST technologies, a new cascaded droplet microfluidic platform that can streamline bacteria/antibiotic mixing, single-cell encapsulation within picoliter droplets, incubation, and detection in a continuous, assembly-line-like workflow is developed. The scalability of the platform is demonstrated by generating 32 groups of ≈10 000 droplets with custom antibiotic conditions within a single device, from which a new statistics-based method is used to analyze the single cell data and produce clinically useful antibiograms with minimum inhibitory concentrations in ≈90 min for the first antibiotic, plus 2 min for each subsequent antibiotic condition. Potential clinical utility of this platform is demonstrated by testing three clinical isolates and eight urine specimens against four frequently used antibiotics, and 100% and 93.8% categorical agreements are achieved compared to laboratory-based results that became available after 48 h.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Bridging the gap between development of point-of-care nucleic acid testing and patient care for sexually transmitted infections},

author = {K. Hsieh, and JH. Melendez and CA. Gaydos and TH. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2022/lc/d1lc00665g},

doi = {DOI	https://doi.org/10.1039/D1LC00665G},

year  = {2021},

date = {2021-11-22},

journal = {Lab on a Chip},

volume = {22},

issue = {3},

pages = {476-511},

abstract = {The incidence rates of sexually transmitted infections (STIs), including the four major curable STIs – chlamydia, gonorrhea, trichomoniasis and, syphilis – continue to increase globally, causing medical cost burden and morbidity especially in low and middle-income countries (LMIC). There have seen significant advances in diagnostic testing, but commercial antigen-based point-of-care tests (POCTs) are often insufficiently sensitive and specific, while near-point-of-care (POC) instruments that can perform sensitive and specific nucleic acid amplification tests (NAATs) are technically complex and expensive, especially for LMIC. Thus, there remains a critical need for NAAT-based STI POCTs that can improve diagnosis and curb the ongoing epidemic. Unfortunately, the development of such POCTs has been challenging due to the gap between researchers developing new technologies and healthcare providers using these technologies. This review aims to bridge this gap. We first present a short introduction of the four major STIs, followed by a discussion on the current landscape of commercial near-POC instruments for the detection of these STIs. We present relevant research toward addressing the gaps in developing NAAT-based STI POCT technologies and supplement this discussion with technologies for HIV and other infectious diseases, which may be adapted for STIs. Additionally, as case studies, we highlight the developmental trajectory of two different POCT technologies, including one approved by the United States Food and Drug Administration (FDA). Finally, we offer our perspectives on future development of NAAT-based STI POCT technologies.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{H.Melendez2021,

title = {Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae using a Phenotypic-Molecular Assay and Lyophilized Antimicrobials},

author = {JH.Melendez and FE. Chen and TH. Wang},

url = {https://www.sciencedirect.com/science/article/pii/S0732889321002819},

doi = {https://doi.org/10.1016/j.diagmicrobio.2021.115590},

year  = {2021},

date = {2021-10-24},

urldate = {2021-10-24},

journal = {Diagnostic Microbiology and Infectious Disease},

volume = {115590},

abstract = {Gonorrhea is an urgent global public health threat as Neisseria gonorrhoeae (Ng) has progressively developed resistance to all antibiotics commonly used for treatment. Surveillance of antimicrobial susceptibility trends is critical to monitor the emergence and spread of antimicrobial resistance. The gold standard methods for antimicrobial susceptibility testing (AST) of Ng are laborious and time-consuming. We evaluated a phenotypic molecular approach, involving a short cultivation step and quantitative PCR, with lyophilized antimicrobials to characterize antimicrobial susceptibility in Ng. There was excellent concordance between AST performed with liquid and lyophilized ciprofloxacin, penicillin, and tetracycline using the pheno-molecular assay, following a four-hour incubation step. The categorical agreement between the pheno-molecular assay and the gold standard AST results was 92.4% for characterization of antimicrobial susceptibility. Essential agreement between the two methods was 91.9%. Characterization of ceftriaxone susceptibility in Ng using the pheno-molecular assay required a six-hour incubation step.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Ying2021,

title = {Methylation-based Cell-free DNA Signature for Early Detection of Pancreatic Cancer},

author = {L. Ying and A. Sharma and A. Chhoda and N. Ruzgar and N. Hasan and R. Kwak and CL. Wolfgang and TH. Wang and JW. Kunstman and RR. Salem and LD. Wood and C. Iacobuzio-Donahue and EB. Schneider and JJ. Farrell and N. Ahuja },

url = {https://pubmed.ncbi.nlm.nih.gov/34860810/},

doi = {DOI: 10.1097/MPA.0000000000001919},

year  = {2021},

date = {2021-10-01},

journal = {Pancreas },

volume = {50},

issue = {9},

pages = {1267-1273},

abstract = {Objectives: The potential of DNA methylation alterations in early pancreatic cancer (PC) detection among pancreatic tissue cell-free DNA seems promising. This study investigates the diagnostic capacity of the 4-gene methylation biomarker panel, which included ADAMTS1, BNC1, LRFN5, and PXDN genes, in a case-control study.



Methods: A genome-wide pharmacoepigenetic approach identified ADAMTS1, BNC1, LRFN5, and PXDN genes as putative targets. Tissue samples including stage I-IV PC (n = 44), pancreatic intraepithelial neoplasia (n = 15), intraductal papillary mucinous neoplasms (n = 24), and normal pancreas (n = 8), and cell-free DNA, which was acquired through methylation on beads technology from PC (n = 22) and control patients (n = 10), were included. The 2-∆ct was the outcome of interest and underwent receiver operating characteristic analysis to determine the diagnostic accuracy of the panel.



Results: Receiver operating characteristic analysis revealed an area under the curve of 0.93 among ADAMTS1, 0.76 among BNC1, 0.75 among PXDN, and 0.69 among LRFN5 gene. The combination gene methylation panel (ADAMTS1, BNC1, LRFN5, and PXDN) had an area under the curve of 0.94, with a sensitivity of 100% and specificity of 90%.



Conclusions: This methylation-based biomarker panel had promising accuracy for PC detection and warranted further validation in prospective PC surveillance trials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {High resolution estimates of relative gene abundance with quantitative ratiometric regression PCR (qRR-PCR)},

author = {A.Y. Trick and F.E. Chen and J.A. Schares and B.E. Freml and P. Lor and Y. Yun and T.H. Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2021/an/d1an01397a},

year  = {2021},

date = {2021-09-30},

journal = {Analyst},

volume = {146},

pages = {6463-6469},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing},

author = {P. Zhang and L. Chen and J. Hu and A.Y. Trick and F.E. Chen and K. Hsieh and Y. Zhao and B. Coleman and K. Kruczynski and T.R Pisanic II and C. D Heaney and W.A, Clarke and Tza-Huei Wang},

url = {https://pubmed.ncbi.nlm.nih.gov/34600203/},

doi = {10.1016/j.bios.2021.113656},

year  = {2021},

date = {2021-09-23},

urldate = {2021-09-23},

journal = {Biosensors and Bioelectronic},

volume = {195},

number = {113656},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {A Rapid Single-Cell Antimicrobial Susceptibility Testing Workflow for Bloodstream Infections},

author = {B. Forsyth and P. Torab and J.H. Lee and T. Malcom and T.H. Wang and J.C. Liao and S.Yang and E. Kvam and C. Puleo and P.K. Wong},

doi = {https://doi.org/10.3390/bios11080288},

year  = {2021},

date = {2021-08-22},

urldate = {2021-08-22},

journal = {Biosensors},

volume = {11},

number = {8},

pages = {288},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2021,

title = {Portable Magnetofluidic Device for Point-of-Need Detection of African Swine Fever},

author = {L. Chen and K. Wen and F.E. Chen and A.Y. Trick and H. Liu and S. Shao and W. Yu and K. Hsieh and Z. Wang and J. Shen and T.H. Wang},

url = {https://pubs.acs.org/doi/full/10.1021/acs.analchem.1c01814},

year  = {2021},

date = {2021-07-28},

journal = {Analytical Chemistry},

volume = {93},

number = {31},

pages = {10940-10946},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2021b,

title = {Point-of-Care CRISPR-Cas-Assisted SARS-CoV-2 Detection in an Automated and Portable Droplet Magnetofluidic Device},

author = { F.E. Chen and P.W. Lee and A.Y. Trick and J.S. Park and L. Chen and K. Shah and H. Mostafa and K.C. Carroll and K. Hsieh and T.H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170879/},

doi = {doi: 10.1016/j.bios.2021.113390},

year  = {2021},

date = {2021-06-02},

journal = {Biosensors and Bioelectronics},

volume = {113390},

abstract = {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 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 leverages facile magnetic concentration and transport of nucleic acid-binding magnetic beads to incorporate sample preparation with minimal manual operation. 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. Finally, 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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {A phase 2 trial of gemcitabine and docetaxel in patients with metastatic colorectal adenocarcinoma with methylated checkpoint with forkhead and ring finger domain promoter and/or microsatellite instability phenotype},

author = {M. Baretti and E. Karunasena and M. Zahurak and R. Walker and Y. Zhao and T.R. Pisanic 2nd and T.H. Wang and T.F. Greten and A.G. Duffy and E. Gootjes and G. Meijer and HMW Verheul and N. Ahuja and J.G. Herman and N.S. Azad},

url = {https://pubmed.ncbi.nlm.nih.gov/33811727/},

doi = {10.1111/cts.12960},

year  = {2021},

date = {2021-05-14},

journal = {Clinical and Translational Science},

volume = {14},

pages = {954-963},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Trick2021,

title = {A portable magnetofluidic platform for detecting sexually transmitted infections and antimicrobial susceptibility},

author = {Alexander Y. Trick and Johan H. Melendez and Fan-En Chen and Liben Chen and Annet Onzia and Aidah Zawedde and Edith Nakku-Joloba and Peter Kyambadde and Emmanuel Mande and Joshua Matovu and Maxine Atuheirwe and Richard Kwizera and Elizabeth A. Gilliams and Yu-Hsiang Hsieh and Charlotte A. Gaydos and Yukari C. Manabe and Matthew M. Hamill and Tza-Huei Wang},

url = {https://stm.sciencemag.org/content/13/593/eabf6356},

doi = {DOI: 10.1126/scitranslmed.abf6356},

year  = {2021},

date = {2021-05-12},

journal = {SCIENCE TRANSLATIONAL MEDICINE},

volume = {13},

number = {593},

pages = {eab6356},

abstract = {Abstract

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.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Zhang2021b,

title = {Facile syringe filter-enabled bacteria separation, enrichment, and buffer exchange for clinical isolation-free digital detection and characterization of bacterial pathogens in urine},

author = {P. Zhang and A. Kaushik and K.E. Mach and K. Hsieh and J.C. Liao and T.H, Wang},

url = {https://pubs.rsc.org/en/content/articlelanding/2021/an/d1an00039j},

year  = {2021},

date = {2021-03-22},

journal = {Analyst},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Kaushik2021,

title = {Droplet-based single-cell measurements of 16S rRNA enable integrated bacteria identification and pheno-molecular antimicrobial susceptibility testing from clinical samples in 30 min},

author = {A.M. Kaushik and K. Hsieh and K.E. Mach and S. Lewis and C.M. Puleo and K.C. Carroll and J.C. Liao and T.H. Wang},

url = {https://onlinelibrary.wiley.com/doi/full/10.1002/advs.202003419},

doi = {https://doi.org/10.1002/advs.202003419},

year  = {2021},

date = {2021-02-01},

journal = {Advanced Science},

volume = {8},

number = {6},

abstract = {Empiric broad‐spectrum antimicrobial treatments of urinary tract infections (UTIs) have contributed to widespread antimicrobial resistance. Clinical adoption of evidence‐based treatments necessitates rapid diagnostic methods for pathogen identification (ID) and antimicrobial susceptibility testing (AST) with minimal sample preparation. In response, a microfluidic droplet‐based platform is developed for achieving both ID and AST from urine samples within 30 min. In this platform, fluorogenic hybridization probes are utilized to detect 16S rRNA from single bacterial cells encapsulated in picoliter droplets, enabling molecular identification of uropathogenic bacteria directly from urine in as little as 16 min. Moreover, in‐droplet single‐bacterial measurements of 16S rRNA provide a surrogate for AST, shortening the exposure time to 10 min for gentamicin and ciprofloxacin. A fully integrated device and screening workflow were developed to test urine specimens for one of seven unique diagnostic outcomes including the presence/absence of Gram‐negative bacteria, molecular ID of the bacteriaas Escherichia coli, an Enterobacterales, or other organism, and assessment of bacterial susceptibility to ciprofloxacin. In a 50‐specimen clinical comparison study, the platform demonstrates excellent performance compared to clinical standard methods (areas‐under‐curves, AUCs >0.95), within a small fraction of the turnaround time, highlighting its clinical utility.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chamling2021,

title = {Single-Cell Transcriptomic Reveals Molecular Diversity and Developmental Heterogeneity of Human Stem Cell-derived Oligodendrocyte Lineage Cells},

author = {X. Chamling and A. Kallman and W. Fang and C. Berlinicke and J. Mertz and P. Devkota and I.M. Pantoja and M. Smith and Z. Ji and C. Chang and A. Kaushik and L. Chen and K. Whartenby and P. Calabresi and H.Q. Mao and H. Ji and T.H. Wang, and D. Zack},

url = {https://www.nature.com/articles/s41467-021-20892-3},

year  = {2021},

date = {2021-01-28},

journal = {Nature Communications},

volume = {12},

number = {652},

abstract = {Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Chen2020,

title = {Toward Decentralizing Antibiotic Susceptibility Testing via Ready-to-Use Microwell Array and Resazurin-Aided Colorimetric Readout},

author = {F.E. Chen and A. Kaushik and K. Hsieh and E. Chang and L. Chen and P. Zhang and T.H. Wang},

url = {https://pubs.acs.org/doi/10.1021/acs.analchem.0c04095},

doi = { https://doi.org/10.1021/acs.analchem.0c04095},

year  = {2021},

date = {2021-01-26},

urldate = {2020-11-29},

journal = {Analytical Chemistry},

volume = {93},

number = {3},

pages = {1260–1265},

abstract = {In the face of the global threat from drug-resistant superbugs, there remains an unmet need for simple and accessible diagnostic tools that can perform important antibiotic susceptibility testing against pathogenic bacteria and guide antibiotic treatments outside of centralized clinical laboratories. As a potential solution to this important problem, we report herein the development of a microwell array-based resazurin-aided colorimetric antibiotic susceptibility test (marcAST). At the core of marcAST is a ready-to-use microwell array device that is preassembled with custom titers of various antibiotics and splits bacterial samples upon a simple syringe injection step to initiate AST against all antibiotics. We also employ resazurin, which changes from blue to pink in the presence of growing bacteria, to accelerate and enable colorimetric readout in our AST. Even with its simplicity, marcAST can accurately measure the minimum inhibitory concentrations of reference bacterial strains against common antibiotics and categorize the antibiotic susceptibilities of clinically isolated bacteria. With more characterization and refinement, we envision that marcAST can become a potentially useful tool for performing AST without trained personnel, laborious procedures, or bulky instruments, thereby decentralizing this important test for combating drug-resistant superbugs.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{Scherer2021,

title = {Digital electrical impedance analysis for single bacterium sensing and antimicrobial susceptibility testing},

author = { B. Scherer and C. Surrette and H. Li, and P. Torab, and E. Kvam and C. Galligan and S. Go and G. Grossmann and T. Hammond and T. Johnson and R. St-Pierre and J. Nelson and R.A. Potyrailo and T. Khire and K. Hsieh and T.H. Wang and P. Kin Wong and C. Puleo },

url = {https://pubs.rsc.org/en/content/articlelanding/2021/lc/d0lc00937g#!divAbstract},

doi = {https://doi.org/10.1039/D0LC00937G},

year  = {2021},

date = {2021-01-26},

journal = {Lab on a Chip},

abstract = {Single-molecule and single-cell analysis techniques have opened new opportunities for characterizing and analyzing heterogeneity within biological samples. These detection methods are often referred to as digital assays because the biological sample is partitioned into many small compartments and each compartment contains a discrete number of targets (e.g. cells). Using digital assays, researchers can precisely detect and quantify individual targets, and this capability has made digital techniques the basis for many modern bioanalytical tools (including digital PCR, single cell RNA sequencing, and digital ELISA). However, digital assays are dominated by optical analysis systems that typically utilize microscopy to analyze partitioned samples. The utility of digital assays may be dramatically enhanced by implementing cost-efficient and portable electrical detection capabilities. Herein, we describe a digital electrical impedance sensing platform that enables direct multiplexed measurement of single cell bacterial cells. We outline our solutions to the challenge of multiplexing impedance sensing across many culture compartments and demonstrate the potential for rapidly differentiating antimicrobial resistant versus susceptible strains of bacteria.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}