Programmable PNA-nanoparticle hybrids as nanoscale recognition architectures for amplification-free nucleic acid recognition.

We have demonstrated that mixed-base PNA oligomers are effective coagulants of citrate ion-coated gold and silver nanoparticles (AuNPs and AgNPs), and PNA-induced particle aggregation can be disrupted by hybridization of PNA with a specific DNA. Using particles' aggregation/dispersion as a measure, we have investigated how PNA and PNA-DNA complexes bind to AuNPs and AgNPs and modulate particles' stability differently relative to their DNA counterparts. We have made the following original discoveries: (1) mix-base PNA oligomers can induce immediate particle aggregation in a concentration- and chain-length-dependent manner; (2) PNA oligomers have a higher affinity to AuNPs and AgNPs than its ssDNA counterpart; (3) PNA-DNA complexes, although having a stable double helix structure similar to dsDNA, can effectively protect the particles from salt induced aggregation, and the protection effect of different nucleic acids are in the order of PNA-DNA complex > ssDNA > dsDNA; (4) all the characteristics are identical for AuNPs and AgNPs; and (5) AgNPs is more sensitive in response to destabilization effect and is proven a more sensitive platform for colorimetric assays. The control of particle aggregation and dispersion by PNA and PNA-DNA complexes has been used to detect a specific DNA sequence with single-base-mismatch resolution. zeta potential measurements have been conducted to reveal how distinct backbone properties of PNA and PNA-DNA complexes relative to their DNA counterparts contribute to the distinct binding characteristics.

In this study, the authors report that sodium citrate can aggregate hexadecyl-trimethyl-ammonium ion(+)-coated gold nanorods (AuNRs), and nucleic acids of different charge and structure properties, i.e., single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded peptide nucleic acid (PNA), and PNA-DNA complex, can bind to the AuNRs and therefore retard the sodium citrate-induced aggregation to different extents. The discovery that hybridized dsDNA (and the PNA-DNA complex) has a more pronounced protection effect than ssDNA (and PNA) allows the authors to develop a homogeneous phase AuNRs-based UV-visible (UV-vis) spectral assay for detecting specific sequences of oligonucleotides (20 mer) with a single-base-mismatch selectivity and a limit of detection of 5 nM. This assay involves no tedious bioconjugation and on-particle hybridization. The simple "set and test" format allows for a highly efficient hybridization in a homogeneous phase and a rapid display of the results in less than a minute. By measuring the degree of reduction in AuNR aggregation in the presence of different nucleic acid samples, one can assess how different nucleic acids interact with the AuNRs to complement the knowledge of spherical gold nanoparticles. Besides UV-vis characterization, transmission electron microscopy and zeta potential measurements were conduced to provide visual evidence of the particle aggregation and to support the discussion of the assay principle.

The crystal structure of a nucleic acid triplex reveals a helix, designated P-form, that differs from previously reported nucleic acid structures. The triplex consists of one polypurine DNA strand complexed to a polypyrimidine hairpin peptide nucleic acid (PNA) and was successfully designed to promote Watson-Crick and Hoogsteen base pairing. The P-form helix is underwound, with a base tilt similar to B-form DNA. The bases are displaced from the helix axis even more than in A-form DNA. Hydrogen bonds between the DNA backbone and the Hoogsteen PNA backbone explain the observation that polypyrimidine PNA sequences form highly stable 2:1 PNA-DNA complexes. This structure expands the number of known stable helical forms that nucleic acids can adopt.

Bioassay development for public health emergency

Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

Nucleic acid detection plays a crucial role in various aspects of health care, necessitating accessible and reliable quantification methods, especially in resource-limited settings. This work presents a simplified electrochemical approach for end-point yet quantitative nucleic acid detection. By elevating the concentration of redox species and choosing potential as the signals, we achieved enhanced signal robustness, even in the presence of interfering substances. Leveraging this robustness, we accurately measured pH-induced redox potential changes in methylene blue solution for end-point nucleic acid detection after loop-mediated isothermal amplification (LAMP). Our method demonstrated quantitative detection of the SARS-CoV-2 N gene and human ATCB gene and successful discrimination of the human BRAF V600E mutation, comparable in sensitivity to commercial kits. The developed user-friendly electrochemical method offers a simplified and reliable approach for end-point yet quantitative detection of nucleic acids, potentially expanding the benefits of nucleic acid testing in resource-limited settings.

Nucleic acid detection technologies have been widely utilized for various diseases. Conventional laboratory tests are less suitable for use in resource-limited settings as they are time-consuming, high-cost, complex, and heavily dependent on benchtop equipment. Rapid nucleic acid detection methods that consist of rapid nucleic acid extraction steps could overcome these challenges. A paper-based platform has been utilized to develop various rapid nucleic acid extraction methods owing to its cost-effectiveness, portability, and easy-modification. However, the existing paper-based nucleic acid extraction technologies mainly focus on improving the adsorption capacity of nucleic acids without reducing the non-specific adsorption capacity of proteins. In this study, paper-based nucleic acid extraction technology with wash-free, elution-free, and low protein adsorption was developed. The fabrication of paper involves the mixing of polyethylene glycol (PEG)-modified cotton fiber, chitosan (COS)-modified cotton fiber, and cotton fiber to form PEG-modified cotton fiber/chitosan-modified cotton fiber/cotton fiber (PEG-CF/COS-CF/CF) paper by the wet molding method. The result showed that PEG-CF/COS-CF/CF paper has a desirable pore size (23.9 ± 4.03 μm), good mechanical strength (dry: 9.37 Mpa and wet: 0.28 Mpa), and hydrophilicity (contact angle: 42.6° ± 0.36°). NH3+ groups of COS and OH- groups of PEG were observed on its surface and the adsorption efficiency of nucleic acid in TE buffer was 42.48% ± 0.30%. The limit of detection of pure DNA with this PEG-CF/COS-CF/CF paper by qPCR was as low as 25 ng. Additionally, this platform could successfully extract nucleic acid from 30 μL of a saliva sample, highlighting its potential use for clinical sample testing. The proposed paper-based nucleic acid extraction platform shows tremendous potential for disease diagnosis in resource-limited settings.

A power-free microfluidic device for the detection of hepatitis B virus in resource-limited settings.

BackgroundHigh quality tests for detection of STIs are available but they are neither affordable nor available to patients in resource-limited settings. For patients presenting with STI symptoms, WHO recommends the...

Visual detection of Mycobacterium tuberculosis in exhaled breath using N95 enrichment respirator, RPA, and lateral flow assay.

The use of point-of-care (POC) devices in limited resource settings where access to commonly used infrastructure, such as water and electricity, can be restricted represents simultaneously one of the best application fits for POC systems as well as one of the most challenging places to deploy them. Of the many challenges involved in these systems, the preparation and processing of complex samples like stool, vomit, and biopsies are particularly difficult due to the high number and varied nature of mechanical and chemical interferents present in the sample. Previously we have demonstrated the ability to use solar-thermal energy to perform PCR based nucleic acid amplifications. In this work demonstrate how the technique, using similar infrastructure, can also be used to perform solar-thermal based sample processing system for extracting and isolating Vibrio Cholerae nucleic acids from fecal samples. The use of opto-thermal energy enables the use of sunlight to drive thermal lysing reactions in large volumes without the need for external electrical power. Using the system demonstrate the ability to reach a 95°C threshold in less than 5 minutes and maintain a stable sample temperature of +/− 2°C following the ramp up. The system is demonstrated to provide linear results between 104 and 108 CFU/mL when the released nucleic acids were quantified via traditional means. Additionally, we couple the sample processing unit with our previously demonstrated solar-thermal PCR and tablet based detection system to demonstrate very low power sample-in-answer-out detection.

Background: Strategies to expand access to routine HIV monitoring assays in resource limited settings (RLS) include the use of point-of-care (POC) tests and dried blood spots (DBS) collected at remote settings and shipped to central laboratories for testing for viral load (VL) testing. Currently access to CD4 testing is gradually expanding due to the emergence of relatively rapid, instrument-based POC tests. There are also instrument-free CD4 tests in the pipeline among which is VISITECT CD4 test, a semi-quantitative lateral-flow based technology. Though few near POC VL tests are commercially available currently and a few in the pipeline, these have not yet had much impact on the total level of VL monitoring in resource-poor settings they. As a result, the use of DBS is suggested as interim option for expanding access to VL testing in RLS. However, due to inclusion of HIV nucleic acids from white blood cells rather than only plasma HIV RNA, many studies reported its lack of accuracy to diagnose anti-retroviral therapy (ART) failure when used in most RT-PCR platforms <br> Aims: The broad aim of this thesis was to enhance access to HIV monitoring assays in RLS by improving performance and utility of assay technologies/methods. Specific aims included; studying the modified use of the standard VISITECT CD4 test for TB/HIV co-infected patients who need a different cut-off for timing of ART initiation, and development of a filter paper-based method of plasma separation (referred to as Filtered and Dried Plasma, FDP) from whole blood to enhance accurate quantification of HIV VL in RLS. <br> Methods: Experiments were conducted to investigate the potential use of test/reference ratio values generated by AX-2 reader of VISITECT CD4 test for TB/HIV co-infected <br> patients ART initiation (Chapter 2). In Chapter 4, different filtration materials (filter papers and biological reagents) and approaches were examined to develop a novel plasma separation method, known as Filtered and Dried Plasma (FDP). Proof-of-concept experiments to evaluate the new method efficiency in removing blood cells and generating cell-free plasma were also conducted. A prospective study to evaluate the performance of FDP in HIV VL quantification was performed using clinical samples (Chapter 5). Furthermore, long term storage stability of FDP was studied in laboratory conditions (Chapter 6). <br> Results: Only preliminary experiments that pinpoint a potential approach where modification of VISITECT CD4 test can be made were conducted and presented in Chapter 2. A preferred method (FDP) to DBS has been developed using filter papers that demonstrated effective elimination of RBCs and 93% of WBCs (Chapter 4). The prospective study (Chapter 5) demonstrated its accurate detection of treatment failure at the current cut-off (1000 cps/ml). Storage stability studies demonstrated adequate stability of FDP both in the pre and post- sample application storage (Chapter 6). <br> Conclusion: This thesis highlighted the diagnostic challenges in HIV/AIDS management, and strategies to enhance access and utility of diagnostics in RLS. Limited work were done and presented under the aim to investigate the modified use of VISITECT CD4 test (Chapter 2) due to delays in the final manufacturing product. The prototype plasma separation method, FDP, demonstrated efficient performance in HIV VL quantification and stability giving the promise to be used as the preferred dried sample for VL testing in RLS.

BackgroundHepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood.MethodsSpectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs.ResultsThe structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs.DiscussionTogether, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

Extraction and concentration of pure nucleic acid from complex biofluids are the prerequisite for nucleic acid amplification test (NAAT) applications in pathogen detection, biowarfare prevention, and genetic diseases. However, conventional spin-column mediated nucleic acid extraction is constricted by the requirement for costly power-intensive centralized lab infrastructure, making it unsuitable for limited-resource settings. Significant progress in lab-on-a-chip devices or cartridges (e.g., Cepheid GeneXpert®) that integrate nucleic acid extraction and amplification has been made, but these approaches either require additional equipment or are costly. Similarly, their complexities make them difficult to fabricate in low-resource settings by the end-user themselves. The application of magnetic particles such as silica-coated iron oxide beads for nucleic acid extraction is relatively instrument-free, rapid, user-friendly, and amenable to automation. But, they rely on hazardous chaotropic salt chemistry and ethanol desalting that could limit their efficacy for downstream NAATs. Recent advances in several types of novel material (e.g., polyamine) coated magnetic bead-based chaotropic salt-free extraction methods offer a possible solution to this problem. However, these materials also involve multistep synthesis impermissible in limited-resource settings. To offer a possible instrument-free magnetic particle-based nucleic acid extraction doable at limited-resource settings, we investigated the nucleic acid capture ability of two chitosan-coated magnetic particles that are preparable by minimally trained personnel using only a water bath and a magnetic stirrer within 6-8 h. We quantitatively probed the efficiency of the passive (without any electrical shaking or vortex-aided) DNA magnetocapture (i.e., binding to chitosan magnetic particles, physical separation from its sample of origin, and release from the particles) using UV260. To explore their suitability towards clinically relevant sensitive downstream NAATs, 100-1000 copies (i.e., in the order of zeptomole) of Escherichia coli (E. coli) or human genomic DNA from aqueous solution, crude cell lysate, and fetal bovine serum were extracted by them and then successfully detected using quantitative real-time loop-mediated isothermal amplification (LAMP) or real-time polymerase chain reaction (PCR). Alongside, their suitability with gel-based LAMP, colorimetric LAMP, and in situ (on beads) LAMP was also probed. The required optimization of the amplification methods has been discussed. Overall, the turnaround time for the magnetocapture combined with NAAT was 1.5-2 h and is thus expected to aid in rapid clinical decision making. With the ease of preparation, reproducibility, and compatibility with downstream NAATs, we anticipate that these magnetic particles would facilitate the expansion and decentralization of nucleic acid-based diagnosis for limited-resource settings.