Fractal circuit sensors enable rapid quantification of biomarkers for donor lung assessment for transplantation.

Biomarker profiling is being rapidly incorporated in many areas of modern medical practice to improve the precision of clinical decision-making. This potential improvement, however, has not been transferred to the practice of organ assessment and transplantation because previously developed gene-profiling techniques require an extended period of time to perform, making them unsuitable in the time-sensitive organ assessment process. We sought to develop a novel class of chip-based sensors that would enable rapid analysis of tissue levels of preimplantation mRNA markers that correlate with the development of primary graft dysfunction (PGD) in recipients after transplant. Using fractal circuit sensors (FraCS), three-dimensional metal structures with large surface areas, we were able to rapidly (<20 min) and reproducibly quantify small differences in the expression of interleukin-6 (IL-6), IL-10, and ATP11B mRNA in donor lung biopsies. A proof-of-concept study using 52 human donor lungs was performed to develop a model that was used to predict, with excellent sensitivity (74%) and specificity (91%), the incidence of PGD for a donor lung. Thus, the FraCS-based approach delivers a key predictive value test that could be applied to enhance transplant patient outcomes. This work provides an important step toward bringing rapid diagnostic mRNA profiling to clinical application in lung transplantation.

Rapid electrochemical phenotypic profiling of antibiotic-resistant bacteria.

Rapid phenotyping of bacteria to identify drug-resistant strains is an important capability for the treatment and management of infectious disease. At present, the rapid determination of antibiotic susceptibility is hindered by the requirement that, in existing devices, bacteria must be pre-cultured for 2-3 days to reach detectable levels. Here we report a novel electrochemical approach that achieves rapid readout of the antibiotic susceptibility profile of a bacterial infection within one hour. The electrochemical reduction of a redox-active molecule is monitored that reports on levels of metabolically-active bacteria. Bacteria are captured in miniaturized wells, incubated with antimicrobials and monitored for resistance. This electrochemical phenotyping approach is effective with clinically-relevant levels of bacteria, and provides results comparable to culture-based analysis. Results, however, are delivered on a much faster timescale, with resistance profiles available after a one hour incubation period.

Nanoparticle-based sorting of circulating tumor cells by epithelial antigen expression during disease progression in an animal model.

Circulating tumor cells (CTCs) can be used as markers for the detection, characterization, and targeted therapeutic management of cancer. We recently developed a nanoparticle-mediated approach for capture and sorting of CTCs based on their specific epithelial phenotype. In the current study, we investigate the phenotypic transition of tumor cells in an animal model and show the correlation of this transition with tumor progression. VX2 tumor cells were injected into rabbits, and CTCs were evaluated during tumor progression and correlated with computerized tomography (CT) measurements of tumor volume. The results showed a dramatic increase of CTCs during the four weeks of tumor growth. Following resection, CTC levels dropped but then rebounded, likely due to lymph node metastases. Additionally, CTCs showed a marked loss of the epithelial cell adhesion molecule (EpCAM) relative to precursor cells. In conclusion, the device accurately traces disease progression and CTC phenotypic shift in an animal model. FROM THE CLINICAL EDITOR: The detection of circulating tumor cells (CTCs) has been used to predict disease prognosis. In this study, the authors developed a nanoparticle-mediated platform based on microfluidics to analyze the differential expressions of epithelial cell adhesion molecule (EpCAM) on CTCs in an animal model. It was found that the loss of EpCAM correlated with disease progression. Hence, the use of this platform may be further applied in other cancer models in the future.

Ultrasensitive visual read-out of nucleic acids using electrocatalytic fluid displacement.

Diagnosis of disease outside of sophisticated laboratories urgently requires low-cost, user-friendly devices. Disposable, instrument-free testing devices are used for home and physician office testing, but are limited in applicability to a small class of highly abundant analytes. Direct, unambiguous visual read-out is an ideal way to deliver a result on a disposable device; however, existing strategies that deliver appropriate sensitivity produce only subtle colour changes. Here we report a new approach, which we term electrocatalytic fluid displacement, where a molecular binding event is transduced into an electrochemical current, which drives the electrodeposition of a metal catalyst. The catalyst promotes bubble formation that displaces a fluid to reveal a high contrast change. We couple the read-out system to a nanostructured microelectrode and demonstrate direct visual detection of 100 fM DNA in 10 min. This represents the lowest limit of detection of nucleic acids reported using high contrast visual read-out.

Velocity valleys enable efficient capture and spatial sorting of nanoparticle-bound cancer cells.

The development of strategies for isolating rare cells from complex matrices like blood is important for a wide variety of applications including the analysis of bloodborne cancer cells, infectious pathogens, and prenatal testing. Due to their high colloidal stability and surface-to-volume ratio, antibody-coated magnetic nanoparticles are excellent labels for cellular surface markers. Unfortunately, capture of nanoparticle-bound cells at practical flow rates is challenging due to the small volume, and thus low magnetic susceptibility, of magnetic nanoparticles. We have developed a means to capture nanoparticle-labeled cells using microstructures which create pockets of locally low linear velocity, termed velocity valleys. Cells that enter a velocity valley slow down momentarily, allowing the magnetic force to overcome the reduced drag force and trap the cells. Here, we describe a model for this mechanism of cell capture and use this model to guide the rational design of a device that efficiently captures rare cells and sorts them according to surface expression in complex matrices with greater than 10,000-fold specificity. By analysing the magnetic and drag forces on a cell, we calculate a threshold linear velocity for capture and relate this to the capture efficiency. We find that the addition of X-shaped microstructures enhances capture efficiency 5-fold compared to circular posts. By tuning the linear velocity, we capture cells with a 100-fold range of surface marker expression with near 100% efficiency and sort these cells into spatially distinct zones. By tuning the flow channel geometry, we reduce non-specific cell adhesion by 5-fold.

Nanoparticle-mediated binning and profiling of heterogeneous circulating tumor cell subpopulations.

The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. CTC capture devices developed to date isolate a bulk population of CTCs and do not differentiate subpopulations that may have varying phenotypes with different levels of clinical relevance. Here, we present a new device for CTC spatial sorting and profiling that sequesters blood-borne tumor cells with different phenotypes into discrete spatial bins. Validation data are presented showing that cancer cell lines with varying surface expression generate different binning profiles within the device. Working with patient blood samples, we obtain profiles that elucidate the heterogeneity of CTC populations present in cancer patients and also report on the status of CTCs within the epithelial-to-mesenchymal transition (EMT).

Ultrasensitive electrochemical biomolecular detection using nanostructured microelectrodes.

Electrochemical sensors have the potential to achieve sensitive, specific, and low-cost detection of biomolecules--a capability that is ever more relevant to the diagnosis and monitored treatment of disease. The development of devices for clinical diagnostics based on electrochemical detection could provide a powerful solution for the routine use of biomarkers in patient treatment and monitoring and may overcome the many issues created by current methods, including the long sample-to-answer times, high cost, and limited prospects for lab-free use of traditional polymerase chain reaction, microarrays, and gene-sequencing technologies. In this Account, we summarize the advances in electrochemical biomolecular detection, focusing on a new and integrated platform that exploits the bottom-up fabrication of multiplexed electrochemical sensors composed of electrodeposited noble metals. We trace the evolution of these sensors from gold nanoelectrode ensembles to nanostructured microelectrodes (NMEs) and discuss the effects of surface morphology and size on assay performance. The development of a novel electrocatalytic assay based on Ru(3+) adsorption and Fe(3+) amplification at the electrode surface as a means to enable ultrasensitive analyte detection is discussed. Electrochemical measurements of changes in hybridization events at the electrode surface are performed using a simple potentiostat, which enables integration into a portable, cost-effective device. We summarize the strategies for proximal sample processing and detection in addition to those that enable high degrees of sensor multiplexing capable of measuring 100 different analytes on a single chip. By evaluating the cost and performance of various sensor substrates, we explore the development of practical lab-on-a-chip prototype devices. By functionalizing the NMEs with capture probes specific to nucleic acid, small molecule, and protein targets, we can successfully detect a wide variety of analytes at clinically relevant concentrations and speeds. Using this platform, we have achieved attomolar detection levels of nucleic acids with overall assay times as short as 2 min. We also describe the adaptation of the sensing platform to allow for the measurement of uncharged analytes--a challenge for reporter systems that rely on the charge of an analyte. Furthermore, the capabilities of this system have been applied to address the many current and important clinical challenges involving the detection of pathogenic species, including both bacterial and viral infections and cancer biomarkers. This novel electrochemical platform, which achieves large molecular-to-electrical amplification by means of its unique redox-cycling readout strategy combined with rapid and efficient analyte capture that is aided by nanostructured microelectrodes, achieves excellent specificity and sensitivity in clinical samples in which analytes are present at low concentrations in complex matrices.

Proximal bacterial lysis and detection in nanoliter wells using electrochemistry.

Rapid and direct genetic analysis of low numbers of bacteria using chip-based sensors is limited by the slow diffusion of mRNA molecules. Long incubation times are required in dilute solutions in order to collect a sufficient number of molecules at the sensor surface to generate a detectable signal. To overcome this barrier here we present an integrated device that leverages electrochemistry-driven lysis less than 50 µm away from electrochemical nucleic acid sensors to overcome this barrier. Released intracellular mRNA can diffuse the short distance to the sensors within minutes, enabling rapid and sensitive detection. We validate this strategy through direct lysis and detection of E. coli mRNA at concentrations as low as 0.4 CFU/µL in 2 min, a clinically relevant combination of speed and sensitivity for a sample-to-answer molecular analysis approach.