skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs
Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, β-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).  more » « less
Award ID(s):
1846740
PAR ID:
10501865
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ;
Publisher / Repository:
Science Advances
Date Published:
Journal Name:
Science Advances
Volume:
9
Issue:
36
ISSN:
2375-2548
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Field deployment is critical to developing numerous sensitive impedance transducers. Precise, cost-effective, and real-time readout units are being sought to interface these sensitive impedance transducers for various clinical or environmental applications. This paper presents a general readout method with a detailed design procedure for interfacing impedance transducers that generate small fractional changes in the impedance characteristics after detection. The emphasis of the design is obtaining a target response resolution considering the accuracy in real-time. An entire readout unit with amplification/filtering and real-time data acquisition and processing using a single microcontroller is proposed. Most important design parameters, such as the signal-to-noise ratio (SNR), common-mode-to-differential conversion, digitization configuration/speed, and the data processing method are discussed here. The studied process can be used as a general guideline to design custom readout units to interface with various developed transducers in the laboratory and verify the performance for field deployment and commercialization. A single frequency readout unit with a target 8-bit resolution to interface differentially placed transducers (e.g., bridge configuration) is designed and implemented. A single MCU is programmed for real-time data acquisition and sine fitting. The 8-bit resolution is achieved even at low SNR levels of roughly 7 dB by setting the component values and fitting algorithm parameters with the given methods. 
    more » « less
  2. Rasmussen, Angela L. (Ed.)
    ABSTRACT Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. IMPORTANCE One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes. 
    more » « less
  3. Abstract Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity. 
    more » « less
  4. null (Ed.)
    Abstract Electronic biosensors for DNA detection typically utilize immobilized oligonucleotide probes on a signal transducer, which outputs an electronic signal when target molecules bind to probes. However, limitation in probe selectivity and variable levels of non-target material in complex biological samples can lead to nonspecific binding and reduced sensitivity. Here we introduce the integration of 2.8 μm paramagnetic beads with DNA fragments. We apply a custom-made microfluidic chip to detect DNA molecules bound to beads by measuring Impedance Peak Response (IPR) at multiple frequencies. Technical and analytical performance was evaluated using beads containing purified Polymerase Chain Reaction (PCR) products of different lengths (157, 300, 613 bp) with DNA concentration ranging from 0.039 amol to 7.8 fmol. Multi-frequency IPR correlated positively with DNA amounts and was used to calculate a DNA quantification score. The minimum DNA amount of a 300 bp fragment coupled on beads that could be robustly detected was 0.0039 fmol (1.54 fg or 4750 copies/bead). Additionally, our approach allowed distinguishing beads with similar molar concentration DNA fragments of different lengths. Using this impedance sensor, purified PCR products could be analyzed within ten minutes to determine DNA fragment length and quantity based on comparison to a known DNA standard. 
    more » « less
  5. Abstract Enzymatic DNA amplification‐based approaches involving intercalating DNA‐binding fluorescent dyes and expensive optical detectors are the gold standard for nucleic acid detection. As components of a simplified and miniaturized system, conventional silicon‐based ion sensitive field effect transistors (ISFETs) that measure a decrease in pH due to the generation of pyrophosphates during DNA amplification have been previously reported. In this article, Bst polymerase in a loop‐mediated isothermal amplification (LAMP) reaction combined with target‐specific primers and crumpled graphene field effect transistors (gFETs) to electrically detect amplification by sensing the reduction in primers is used. Graphene is known to adsorb single‐stranded DNA due to noncovalent π–π bonds, but not double‐stranded DNA. This approach does not require any surface functionalization and allows the detection of primer concentrations at the endpoint of reactions. As recently demonstrated, the crumpled gFET over the conventional flat gFET sensors due to their superior sensitivity is chosen. The endpoint of amplification reaction with starting concentrations down to 8 × 10−21min 90 min including the time of amplification and detection is detected. With its high sensitivity and small footprint, this platform will help bring complex lab‐based diagnostic and genotyping amplification assays to the point‐of‐care. 
    more » « less