New Trimodal Phenotypic Reporter of Extended-Spectrum ß-Lactamase Activity.

Bacterial resistance to ß-lactam antibiotics continues to grow as misadministration presents evolutionary pressure that drives bacteria to develop improved resistance enzymes. Known as extended-spectrum ß-lactamases (ESBLs), these enzymes are capable of hydrolyzing advanced ß-lactam antibiotics such as third-generation (and higher) cephalosporins. Phenotypic detection substrates can be used to rapidly identify a cultured patient sample prior to confirmation by more exhaustive but slower means, critically aiding in the antibiotic stewardship essential in maintaining the effectiveness of not only the cephalosporins but also indirectly the carbapenems, our last-resort ß-lactams. To enhance the phenotypic detection arsenal, we have designed an ESBL detection substrate that releases a glucose molecule upon ß-lactamase hydrolysis. Because many forms of detection for glucose exist, the substrate enables ESBL quantification via three modalities commonly found in the clinical laboratory: optical absorbance, for use with the most common microbiology platforms; fluorescence, for enhanced sensitivity; and electrochemistry, which offers the potential for integration into a hand-held platform similar to a personal glucometer. Moreover, we demonstrate that, as opposed to currently available phenotypic detection substrates, our new substrate is engineered to be resistant to older and narrower ß-lactamases, thus enabling specific identification of newer and more dangerous ESBLs.

Demonstration of a quantitative triplex LAMP assay with an improved probe-based readout for the detection of MRSA.

As molecular diagnostics move away from polymerase chain reaction (PCR) in order to target point-of-care testing applications, loop-mediated isothermal amplification (LAMP) is gaining popularity due its rapid, sensitive and specific detection with simpler instrumentation. However, while Taqman PCR enables real-time quantitative readout and multiplexed gene detection in single samples, analogous methods in LAMP are not yet broadly developed. To date, the real-time detection methods applied to LAMP involve turbidimetry or measuring fluorescence of an intercalator; however, both of these methods are nonspecific to the target of interest and do not allow for multiple gene detection in a single sample. Probe-based methods have been developed to address the need for specific target detection and multiplexed, one-pot reactions, but most of these methods have strict assay conditions and require the design of loop primers, which is not always possible. DARQ LAMP is a probe-based method that offers the most promise for quantitative and real-time multiplexed detection, as it has a relatively simple design and can be used in either a four-primer or six-primer system. However, previous work has only shown the assay to function well in a narrow range of reaction conditions, which is restrictive given that various LAMP assays require a broad range of conditions. In this work we investigate the use of the newest-generation strand-displacing polymerase and demonstrate that it has higher tolerance to reaction conditions than previous polymerases. Using the results from these studies, we demonstrate a single-reaction triplex assay for the detection of methicillin-resistant S. aureus (MRSA), which would not be possible with any of the previously reported LAMP systems.

Simplifying Nucleic Acid Amplification from Whole Blood with Direct Polymerase Chain Reaction on Chitosan Microparticles.

Tremendous advances have been made in the development of portable nucleic acid amplification devices for near-patient use. However, the true limitation in the realization of nucleic acid amplification tests (NAATs) for near-patient applications is not the amplification reaction, it is the complexity of the sample preparation. Conventional approaches require several precise intervention steps during the protocol. There are numerous reports in the literature that mimic the sample preparation procedure within a lab-on-a-chip device or cartridge, but these systems require a high number of integrated steps, making the devices and/or their supporting equipment too complex to meet the necessary cost targets and regulatory requirements for near-patient applications. Here we report a simplified method to purify and amplify DNA from complex samples in a minimal number of steps. We show that chitosan-coated microparticles can lyse human cells and capture the released DNA in a single mechanical agitation step, and we show that bound DNA can be amplified directly from the microparticle surface when the magnetic microparticles are transferred to a polymerase chain reaction (PCR). This procedure eliminates (i) the use of PCR-inhibiting reagents (e.g., chaotropic salts and alcohol) and (ii) the washing and elution steps that are required to remove these reagents and release DNA in typical NAAT sample preparation methods. To illustrate the use of this direct PCR method in diagnostics, we amplify human genomic DNA sequences from a ∼1 µL droplet of whole blood, and we amplify plasmid DNA spiked into whole blood droplets to represent circulating viral DNA or cell-free DNA. The qPCR threshold cycle for direct PCR from whole blood is comparable to that of direct PCR with purified DNA, demonstrating that the lysis and capture steps effectively bind DNA and sufficiently enable its amplification. Furthermore, the efficient amplification of plasmid DNA spiked into whole blood proves that the large mass of human genomic DNA captured from the lysed cells does not inhibit the capture and amplification of other circulating DNA. We anticipate that this new streamlined method for preparing DNA for amplification will expand the diagnostic applications of nucleic acid amplification tests, in particular for near-patient applications.

Capture and Direct Amplification of DNA on Chitosan Microparticles in a Single PCR-Optimal Solution.

While nucleic acid amplification tests have great potential as tools for rapid diagnostics, complicated sample preparation requirements inhibit their use in near-patient diagnostics and low-resource-setting applications. Recent advancements in nucleic acid purification have leveraged pH-modulated charge switching polymers to reduce the number of steps required for sample preparation. The polycation chitosan (pKa 6.4) has been used to efficiently purify DNA by binding nucleic acids in acidic buffers and then eluting them at a pH higher than 8.0. Though it is an improvement over conventional methods, this multistep procedure has not transformed the application of nucleic acid amplification assays. Here we describe a simpler approach using magnetic chitosan microparticles that interact with DNA in a manner that has not been reported before. The microparticles capture DNA at a pH optimal for PCR (8.5) just as efficiently as at low pH. Importantly, the captured DNA is still accessible by polymerase, enabling direct amplification from the microparticles. We demonstrate quantitative PCR from DNA captured on the microparticles, thus eliminating nearly all of the sample preparation steps. We anticipate that this new streamlined method for preparing DNA for amplification will greatly expand the diagnostic applications of nucleic acid amplification tests.

Cavity-induced microstreaming for simultaneous on-chip pumping and size-based separation of cells and particles.

We present a microfluidic platform for simultaneous on-chip pumping and size-based separation of cells and particles without external fluidic control systems required for most existing platforms. The device utilizes an array of acoustically actuated air/liquid interfaces generated using dead-end side channels termed Lateral Cavity Acoustic Transducers (LCATs). The oscillating interfaces generate local streaming flow while the angle of the LCATs relative to the main channel generates a global bulk flow from the inlet to the outlet. The interaction of these two competing velocity fields (i.e. global bulk velocity vs. local streaming velocity) is responsible for the observed separation. It is shown that the separation of 5 µm and 10 µm polystyrene beads is dependent on the ratio of these two competing velocity fields. The experimental and simulation results suggest that particle trajectories based only on Stokes drag force cannot fully explain the separation behavior and that the impact of additional forces due to the oscillating flow field must be considered to determine the trajectory of the beads and ultimately the separation behavior of the device. To demonstrate an application of this separation platform with cellular components, smaller red blood cells (7.5 ± 0.8 µm) are separated from larger K562 cells (16.3 ± 2.0 µm) with viabilities comparable to those of controls based on a trypan blue exclusion assay.